Desaturase genes, enzymes encoded thereby, and uses thereof

ABSTRACT

Disclosed are isolated polynucleotides encoding an omega-3 desaturase and a delta-12 desaturase, the enzymes encoded by the isolated polynucleotides, vectors containing the isolated polynucleotides, transgenic hosts that contain the isolated polynucleotides that express the enzymes encoded thereby, methods for producing the desaturase enzymes, and method of using the enzymes to make polyunsaturated fatty acids. The isolated polynucleotides are derived from a fungus,  Saprolegnia diclina  (ATCC 56851). In particular, omega-3-desaturase may be utilized, for example, in the conversion of arachidonic acid (AA) to eicosapentaenoic acid (EPA). Delta-12 desaturase may be used, for example, in the conversion of oleic acid (OA) to linoleic (LA). EPA or polyunsaturated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

FIELD OF THE INVENTION

[0001] The present invention is directed to the identification andisolation of novel genes that encode enzymes involved in the synthesisof polyunsaturated fatty acids (PUFAs). The invention is also directedto the novel desaturase enzymes encoded by these genes and to methods ofutilizing the genes and/or the enzymes encoded by the genes genes. Inparticular, the invention is directed to genes derived from the fungusSaprolegnia diclina (ATCC 56851) that encode a novel ω3-desaturase (alsoreferred to herein as a Δ17-desaturase) and a novel Δ12-desaturase.These enzymes catalyze the introduction of a carbon-carbon double bondbetween a particular position within a fatty acid substrate. Forexample, the novel ω3-desaturase disclosed herein catalyzes theconversion of arachidonic acid (20:4n-6) to eicosapentaenoic acid(20:5n-3) (as well as other desaturation reactions involving othersubstrates). Likewise, the novel Δ12-desaturase disclosed hereincatalyzes the conversion of oleic acid (18:1n-9) to linoleic acid(18:2n-6). The PUFAs so formed may be added to pharmaceuticalcompositions, nutritional compositions, animal feeds, or other products.

BACKGROUND

[0002] Desaturases are a class of enzymes critical in the production oflong-chain polyunsaturated fatty acids. Polyunsaturated fatty acids(PUFAs) play many roles in the proper functioning of all life forms. Forexample, PUFAs are important components of the plasma membrane of acell, where they are found in the form of phospholipids. PUFAs also areprecursors to mammalian prostacyclins, eicosanoids, leukotrienes andprostaglandins. Additionally, PUFAs are necessary for the properdevelopment of the infant brain, as well as for tissue formation andrepair in mature mammals. In view of the biological significance ofPUFAs, attempts are being made to produce them in an efficient manner.

[0003] A number of enzymes, most notably desaturases and elongases, areinvolved in PUFA biosynthesis (see FIG. 1). Elongases catalyze theaddition of a 2-carbon unit to a fatty acid substrate. Thus, forexample, an elongase (generically designated “elo” in FIG. 1) catalyzesthe conversion of γ-linolenic acid (18:3n-6) to dihomo-γ-linolenic acid(20:3n-6), as well as the conversion of stearidonic acid (18:4n-3) toeicosatetraenoic acid (20:4n-3), etc.

[0004] Desaturases catalyze the introduction of unsaturations (i.e.,double bonds) between carbon atoms within the fatty acid alkyl chain ofthe substrate. Thus, for example, linoleic acid (18:2n-6) is producedfrom oleic acid (18:1n-9) by the action of a Δ12-desaturase. Similarly,γ-linolenic acid (18:3n-6) is produced from linoleic acid by the actionof a Δ6-desaturase.

[0005] Throughout the present application, PUFAs will be unambiguouslyidentified using the “omega” system of nomenclature favored byphysiologists and biochemists, as opposed to the “delta” system orI.U.P.A.C. system normally favored by chemists. In the “omega” system, aPUFA is identified by a numeric designation of the number of carbons inthe chain. This is followed by a colon and then another numericdesignation of the number of unsaturations in the molecule. This is thenfollowed by the designation “n-x,” where x is the number of carbons fromthe methyl end of the molecule where the first unsaturation is located.Each subsequence unsaturation (where there is more than one double bond)is located 3 addition carbon atoms toward the carboxyl end of themolecule. Thus, the PUFAs described herein can be described as being“methylene-interrupted” PUFAs. Where some other designation is required,deviations from the “omega” system will be noted.

[0006] Where appropriate, the action of the desaturase enzymes describedherein will also be identified using the “omega” system. Thus, an“omega-3” desaturase catalyzes the introduction of a double bond betweenthe two carbons at positions 3 and 4 from the methyl end of thesubstrate. However, in many instances, it is more convenient to indicatethe activity of a desaturase by counting from the carboxyl end of thesubstrate. Thus, as shown in FIG. 1, a Δ9-desaturase catalyzes theintroduction of a double bond between the two carbons at positions 9 and10 from the carboxyl end of the substrate. In short, where the term“omega” is used, the position on the substrate is being designatedrelative to the methyl terminus; where the term “delta” is used, theposition on the substrate is being designated relative to the carboxylterminus.

[0007] It must be noted that mammals cannot desaturate fatty acidsubstrates beyond the Δ9 position (i.e., beyond 9 carbon atoms distantfrom the carboxyl terminus). Thus, for example, mammals cannot convertoleic acid (18:1n-9) into linoleic acid (18:2n-6); linoleic acidcontains an unsaturation at position Δ12. Likewise, a-linolenic acid(18:3n-3)(having unsaturations at Δ12 and Δ15) cannot be synthesized bymammals. However, for example, mammals can convert α-linolenic acid intostearidonic acid (18:4n-3) by the action of a Δ6-desaturase. (SeeFIG. 1. See also PCT publication WO 96/13591; The FASEB Journal,Abstracts, Part I, Abstract 3093, page A532 (Experimental Biology 98,San Francisco, Calif., Apr. 18-22, 1998); and U.S. Pat. No. 5,552,306.)

[0008] Still referring to FIG. 1, in mammals, fungi, and algae, thestearidonic acid so formed is converted into eicosatetraenoic acid(20:4n-3) by the action of an elongase. This PUFA can then be convertedto eicosapentaenoic acid (20:5n-3) by a Δ5-desaturase. Eicosapentaenoicacid can then, in turn, be converted to ω3-docosapentaenoic acid(22:5n-3) by an elongase.

[0009] Other eukaryotes, including fungi and plants, have enzymes thatdesaturate fatty acid substrates at carbon Δ12 (see PCT publication WO94/11516 and U.S. Pat. No. 5,443,974) and at carbon delta-15 (see PCTpublication WO 93/11245). The major polyunsaturated fatty acids ofanimals therefore are either derived from diet and/or from desaturationand elongation of linoleic acid or α-linolenic acid. In view of thesedifficulties, there remains a significant need to isolate genes involvedin PUFA synthesis. Ideally, these genes would originate from speciesthat naturally produce fatty acids that are not produced naturally inmammals. These genes could then be expressed in a microbial, plant, oranimal system, which would thereby be altered to produce commercialquantities of one or more PUFAs. Thus, there is a definite need fornovel Δ12- and Δ17-desaturase enzymes, the respective genes encodingthese enzymes, as well as recombinant methods of producing theseenzymes. Additionally, a need exists for oils containing levels of PUFAsbeyond those naturally present. Access to such Δ12- and Δ17-desaturaseenzymes allows for the production of large amounts of PUFAs that cannotbe synthesized de novo in mammals. These PUFAs can be used aspharmaceutical agents and/or nutritional supplements.

[0010] All patents, patent publications and priority documents citedherein are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0011] One embodiment of the present invention encompasses an isolatednucleotide acid sequence or fragment thereof comprising or complementaryto a nucleotide sequence encoding a polypeptide having desaturaseactivity, wherein the amino acid sequence of the polypeptide has atleast 50% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:26 and SEQ ID NO:42.

[0012] The present invention also includes an isolated nucleotidesequence (or fragment thereof) comprising or complementary to at least50% of the nucleotide sequence selected from the group consisting of SEQID NO:25 and SEQ ID NO:41. In particular, the sequence may be selectedfrom the group consisting of SEQ ID NO:25 and SEQ ID NO:41. The sequencemay encode a functionally active desaturase which utilizes apolyunsaturated fatty acid as a substrate. Furthermore, the nucleotidesequence may be isolated from a fungus, such as Saprolegnia diclina.

[0013] An additional embodiment of the present invention includes apurified polypeptide encoded by the nucleotide sequences describedabove.

[0014] The present invention also includes a purified polypeptide thatdesaturates a polyunsaturated fatty acid substrate at an omega-3 carbonof the substrate and has at least 50% amino acid identity to an aminoacid sequence comprising SEQ ID NO: 26. The Polypeptide may desaturate afatty acid substrate having 20 carbon atoms.

[0015] Additionally, the present invention encompasses a purifiedpolypeptide that desaturates a polyunsaturated fatty acid substrate at adelta-12 carbon of the substrate and has at least 50% amino acididentity to SEQ ID NO: 42. The polypeptide may desaturate a fatty acidsubstrate having 18 carbon atoms.

[0016] Another embodiment of the present invention includes a method ofproducing a desaturase comprising the steps of: isolating a nucleotidesequence comprising or complementary to at least 50% of the nucleotidesequence selected from the group consisting of SEQ ID NO: 25 and SEQ IDNO: 41; constructing a vector comprising the isolated nucleotidesequence; and introducing the vector into a host cell for a time andunder conditions sufficient for expression of a desaturase encoded bythe isolated nucleotide sequence.

[0017] A further embodiment of the present invention includes a vectorcomprising: 1) an isolated nucleotide sequence corresponding to orcomplementary to at least about 50% of the nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 25 and SEQ ID NO: 41, operablylinked to b) a regulatory sequence.

[0018] Additionally, another embodiment of the present inventionincludes a host cell comprising the above vector. The host cell may be,for example, a eukaryotic cell selected from the group consisting of amammalian cell, an insect cell, a plant cell and a fungal cell. Withrespect to the host cell, expression of the isolated nucleotide sequenceof the vector may result in the host cell producing a polyunsaturatedfatty acid that is not produced in a wild-type of the host cell.

[0019] Also, the present invention encompasses a plant cell, plant, orplant tissue comprising the vector described above, wherein expressionof the nucleotide sequence of the vector results in production of apolyunsaturated fatty acid by the plant cell, plant or plant tissue. Thevector in the plant cell, plant or plant tissue may induce theproduction of a polyunsaturated fatty acid selected from the groupconsisting of, for example, linoleic acid, eicosatetraenoic acid andeicosapentaenoic acid. Also, the invention includes one or more plantoils or acids expressed by the plant cell, plant or plant tissue.

[0020] The invention also includes a transgenic plant comprising theabove-described vector, wherein expression of the nucleotide sequence ofthe vector results in production of a polyunsaturated fatty acid inseeds of the transgenic plant.

[0021] Another embodiment of the present invention encompasses a methodfor producing a polyunsaturated fatty acid comprising the steps of:isolating a nucleotide sequence comprising or complementary to at leastabout 50% of the nucleotide sequence selected from the group consistingof SEQ ID NO: 25 and SEQ ID NO: 41; constructing a vector comprising theisolated nucleotide sequence; transforming the vector into a host cellfor a time and under conditions sufficient for expression of adesaturase encoded by the isolated nucleotide sequence; and exposing theexpressed desaturase selected from the group consisting of anomega-3-desaturase and a delta 12-desaturase, to a fatty acid substrate,whereby the substrate is catalytically converted by said desaturase intoa desired polyunsaturated fatty acid product. The substrate isdihomo-gamma-linolenic acid or arachidonic acid and the productpolyunsaturated fatty acid is eicosatetraenoic acid or eicosapentaenoicacid, respectively, when the expressed desaturase is anomega-3-desaturase. The substrate polyunsaturated fatty acid is oleicacid and the product polyunsaturated fatty acid is linoleic acid, whenthe expressed desaturase is a delta 12-desaturase.

[0022] The method may further comprise the step of exposing thepolyunsaturated fatty acid product to one or more enzymes selected fromthe group consisting of a desaturase and an elongase, whereby thepolyunsaturated fatty acid product is catalytically converted intoanother polyunsaturated fatty acid product. The product polyunsaturatedfatty acid is eicosatetraenoic acid or eicosapentaenoic acid and theanother polyunsaturated fatty acid is eicosapentaenoic acid or omega3-docosapentaenoic acid, respectively, when the expressed desaturase isan omega 3-desaturase. The product polyunsaturated fatty acid islinoleic acid and the another polyunsaturated fatty acid isgamma-linolenic acid, when the expressed desaturase is a delta12-desaturase.

[0023] Additionally, the method described directly above may furthercomprise the step of exposing the another polyunsaturated fatty acid toone or more enzymes selected from the group consisting of a desaturaseand an elongase in order to convert the another polyunsaturated fattyacid to a final polyunsaturated fatty acid. The final polyunsaturatedfatty acid is selected from the group consisting of omega3-docosapentaenoic acid and docosahexaenoic acid, when the expresseddesaturase of step (d) is an omega 3-desaturase. In contrast, the finalpolyunsaturated fatty acid is selected from the group consisting ofdihomo-gamma-linolenic acid, arachidonic acid, adrenic acid, omega6-docosapentaenoic acid, eicosatetraenoic acid, stearidonic acid,eicosapentaenoic acid, omega 3-docosapentaenoic acid and docosahexaenoicacid, when the expressed desaturase is a delta 12-desaturase.

[0024] An additional embodiment of the present invention includes amethod of producing a polyunsaturated fatty acid comprising exposing afatty acid substrate to a polypeptide having at least 50% amino acididentity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 26 and SEQ ID NO: 42, whereby the fatty acid substrate iscatalytically converted into the polyunsaturated fatty acid product. Thefatty acid substrate is dihomo-gamma-linolenic acid or arachidonic acidand the product polyunsaturated fatty acid is eicosatetraenoic acid oreicosapentaenoic acid, respectively, when the polypeptide is an omega3-desaturase. In contrast, the fatty acid substrate is oleic acid andthe polyunsaturated fatty acid product is linoleic acid, when thepolypeptide is a delta 12-desaturase.

[0025] A further embodiment of the present invention includes acomposition comprising at least one polyunsaturated fatty acid selectedfrom the group consisting of the “product” polyunsaturated fatty acidproduced according to the method described above, the “another”polyunsaturated fatty acid produced according to the method describedabove, and the “final” polyunsaturated fatty acid produced according tothe method described above. The product polyunsaturated fatty acid iseicosatetraenoic acid or eicosapentaenoic acid, when the expresseddesaturase of is an omega 3-desaturase. In contrast, the productpolyunsaturated fatty acid is linoleic acid, when the expresseddesaturase is a delta 12-desaturase. The another polyunsaturated fattyacid is eicosapentaenoic acid or omega 3-docosapentaenoic acid,respectively, when the expressed desaturase is an omega 3-desaturase.However, the another polyunsaturated fatty acid is gamma-linolenic acid,when the expressed desaturase is a delta 12-desaturase. The finalpolyunsaturated fatty acid is selected from the group consisting ofomega 3-docosapentaenoic acid and docosahexaenoic acid, when theexpressed desaturase is an omega 3-desaturase. In contrast, the finalpolyunsaturated fatty acid is selected from the group consisting ofdihomo-gamma-linolenic acid, arachidonic acid, adrenic acid, omega6-docosapentaenoic acid, eicosatetraenoic acid, stearidonic acid,eicosapentaenoic acid, omega 3-docosapentaenoic acid and docosahexaenoicacid, when the expressed desaturase is a delta 12-desaturase.

[0026] A further embodiment of the present invention includes a methodof preventing or treating a condition caused by insufficient intake ofat least one polyunsaturated fatty acid comprising administering to thepatient the above-described composition in an amount sufficient toeffect the prevention or treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a schematic showing the biosynthetic pathway leading tothe production of various PUFAs.

[0028]FIG. 2 is the nucleotide sequence of sdd17 (SEQ ID NO: 25), a genederived from S. diclina (ATCC 56851) that encodes a novel ω3-fatty aciddesaturase.

[0029]FIG. 3 is the amino acid sequence of the ω3-desaturase (SDD17)(SEQ ID NO: 26) encoded by the nucleotide sequence depicted in FIG. 2.

[0030]FIG. 4 is an amino acid sequence comparison between the SDD17desaturase depicted in FIG. 3 and a known Δ15-desaturase fromSynechocystis sp. (SYCDESB).

[0031]FIG. 5 is an amino acid sequence comparison between the SDD17desaturase depicted in FIG. 3 and a known Δ17-desaturase from C. elegans(CELEFAT).

[0032]FIG. 6 is the nucleotide sequence of sdd12 (SEQ ID NO:41), a genederived from S. diclina (ATCC 56851) that encodes a novel Δ12-fatty aciddesaturase.

[0033]FIG. 7 is the amino acid sequence of the Δ12-desaturase (SDD12)(SEQ ID NO: 42) encoded by the nucleotide sequence depicted in FIG. 6.

[0034]FIG. 8 is an amino acid sequence comparison between the SDD12desaturase depicted in FIG. 7 and a known Δ12-desaturase from G.hirsutum (GHO6DES).

[0035]FIG. 9 lists the sequence identifiers used throughout theapplication as well as the corresponding amino acid or nucleotidesequence.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Abbreviations Utilized Herein:

[0037] 18:1n-9=oleic acid=OA

[0038] 18:2n-6=linoleic acid=LA

[0039] 18:3n-6=gamma-linolenic acid=GLA

[0040] 18:3n-3=alpha-linolenic acid=ALA

[0041] 18:4n-3=stearidonic acid=STA

[0042] 20:3n-6=dihomo-gamma-linolenic acid=DGLA

[0043] 20:4n-6=arachidonic acid=AA

[0044] 20:4n-3=eicosatetraenoic acid=ETA

[0045] 20:5n-3=eicosapentaenoic acid=EPA

[0046] 22:4n-6=adrenic acid

[0047] 22:5n-3=omega-3-docosapentaenoic acid=DPA

[0048] 22:6n-3=docosahexaenoic acid=DHA

[0049] PUFA=polyunsaturated fatty acid

[0050] The subject invention relates to the nucleotide and translatedamino acid sequences of the ω3-desaturase and Δ12-desaturase genesisolated from the fungus Saprolegnia diclina or S. diclina (ATCC 56851).Furthermore, the subject invention also includes uses of these genes andof the enzymes encoded by these genes. For example, the genes and theircorresponding enzymes may be used in the production of polyunsaturatedfatty acids such as linoleic acid, eicosapentaenoic acid, and the like.These fatty acids can be added to pharmaceutical compositions,nutritional compositions, and to other valuable products.

[0051] The fungus S. diclina (ATCC 56851), from which thepolynucleotides described herein were isolated, is availablecommercially from the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110. The fungus is supplied frozen and can bepropagated in ATCC medium 307 cornmeal agar (Difco #0386) at 24° C. Forfurther information on this fungus, see Beakes G. (1983) “A comparativeaccount of cyst coat ontogeny in saprophytic and fish-lesion(pathogenic) isolates of the Saprolegnia diclina-parasitica complex.”Can. J. Bot. 61, 603-625; and Willoughby L. G., et al. (1983) “Zoosporegermination of Saprolegnia pathogenic to fish.” Trans. Br. Mycol. Soc.80, 421-435.

[0052] The ω3-Desaturase Gene, the Δ12-Desaturase Gene, and the EnzymesEncoded Thereby

[0053] The enzymes encoded by the omega-3 desaturase and delta-12desaturase genes of the present invention are essential in theproduction of PUFAs having at least two unsaturations and an overalllength of 18 carbons or longer. The nucleotide sequence of the isolatedSaprolegnia diclina omega-3 desaturase gene is shown in SEQ ID NO: 25and in FIG. 2. This gene differs significantly in sequence from allknown desaturase genes, from any source. The encoded omega-3 desaturaseenzyme is shown in SEQ ID NO: 26 and in FIG. 3. The nucleotide sequenceof the isolated Saprolegnia diclina delta-12 desaturase gene is shown inSEQ ID NO: 41 and in FIG. 6. This gene also differs significantly insequence from all known desaturase genes, from any source. The encodeddelta-12 desaturase enzyme is shown in SEQ ID NO: 42 and in FIG. 7.

[0054] The isolated omega-3 desaturase gene of the present invention,when transformed into a yeast host, produces an omega-3 desaturaseenzyme that readily catalyzes the conversion of DGLA to ETA, AA to EPA,and adrenic acid to DPA (see Example 5). In like manner, the isolateddelta-12 desaturase gene of the present invention, when transformed intoa yeast host, produces a delta-12 desaturase enzyme that readilycatalyzes the conversion OA to LA (see Example 9).

[0055] It should be noted that the present invention also encompassesnucleotide sequences (and the corresponding encoded proteins) havingsequences comprising, identical to, or complementary to at least about50%, preferably at least about 60%, and more preferably at least about70%, even more preferably at least about 80%, and most preferably atleast about 90% of the nucleotides (i.e., having sequence identity tothe sequence) shown in SEQ ID NO: 25 and SEQ ID NO: 41 (i.e., thenucleotide sequences of the omega-3 desaturase gene and the delta-12desaturase gene of Saprolegnia diclina, respectively) described herein.(All integers between 50% and 100% are also considered to be within thescope of the present invention with respect to percent identity.) Suchsequences may be derived from any source, either isolated from a naturalsource, or produced via a semi-synthetic route, or synthesized de novo.Such sequences may be isolated from or derived from fungal sources, aswell as other non-fungal sources, such as bacterial, algal, C. elegans,mouse or human.

[0056] The present invention also encompasses fragments and derivativesof the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 41, aswell as fragments and derivatives of the sequences derived from othersources, and having the above-described complementarity, identity orcorrespondence. Functional equivalents of the above-sequences (i.e.,sequences having omega-3 desaturase activity or delta-12 desaturaseactivity, as appropriate) are also encompassed by the present invention.

[0057] For purposes of the present invention, a “fragment” of anucleotide sequence is defined as a contiguous sequence of approximatelyat least 6, preferably at least about 8, more preferably at least about10 nucleotides, and even more preferably at least about 15 nucleotidescorresponding to a region of the specified nucleotide sequence.

[0058] Sequence identity or percent identity is the number of exactmatches between two aligned sequences divided by the length of theshorter sequence and multiplied by 100. An approximate alignment fornucleic acid sequences is provided by the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2:482-489 (1981).This algorithm may be extended to use with peptide or protein sequencesusing the scoring matrix created by Dayhoff, Atlas of Protein Sequencesand Structure, M. O. Dayhoff ed., 5 Suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-66763 (1986). The GeneticsComputer Group (GCG) (Madison, Wis.) provides a computer program thatautomates this algorithm for both nucleic acid and peptide sequences inthe “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from GCG). Other equally suitableprograms for calculating the percent identity or similarity betweensequences are generally known in the art.

[0059] The invention also includes a purified polypeptide whichdesaturates PUFAs at the omega-3 position and has at least about 50%amino acid similarity or identity, preferably at least about 60%similarity or identity, more preferably at least about 70% similarity oridentity, even more preferably at least about 80% similarity oridentity, and most preferably at least about 90% similarity or identityto the amino acid sequence shown in SEQ ID NO: 26 (FIG. 3) and encodedby the above-noted nucleotide sequence(s) (All integers between 50% and100% similarity or identity are also included within the scope of theinvention.) The invention further includes a purified polypeptide whichdesaturates PUFAs at the delta-12 position and has at least about 50%amino acid similarity or identity, preferably at least about 60%similarity or identity, more preferably at least about 70% similarity oridentity, even more preferably at least about 80% similarity oridentity, and most preferably at least about 90% similarity or identityto the amino acid sequence shown in SEQ. ID. NO: 42 (FIG. 7) which, inturn, is encoded by the above-described nucleotide sequence(s). (Allintegers between 50% and 100% similarity or identity are also includedwithin the scope of the invention.)

[0060] The term “identity” refers to the relatedness of two sequences ona nucleotide-by-nucleotide basis over a particular comparison window orsegment. Thus, identity is defined as the degree of sameness,correspondence or equivalence between the same strands (either sense orantisense) of two DNA segments. “Percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over aparticular region, determining the number of positions at which theidentical base occurs in both sequence in order to yield the number ofmatched positions, dividing the number of such positions by the totalnumber of positions in the segment being compared and multiplying theresult by 100. Optimal alignment of sequences may be conducted by thealgorithm of Smith & Waterman, Appl. Math. 2:482 (1981), by thealgorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by themethod of Pearson & Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988)and by computer programs which implement the relevant algorithms (e.g.,Clustal Macaw Pileup(http://cmgm.stanford.edu/biochem218/11Multiple.pdf; Higgins et al.,CABIOS. 5L151-153 (1989)), FASTDB (Intelligenetics), BLAST (NationalCenter for Biomedical Information; Altschul et al., Nucleic AcidsResearch 25:3389-3402 (1997)), PILEUP (Genetics Computer Group, Madison,Wis.) or GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, Madison, Wis.). (See U.S.Pat. No. 5,912,120.)

[0061] For purposes of the present invention, “complementarity isdefined as the degree of relatedness between two DNA segments. It isdetermined by measuring the ability of the sense strand of one DNAsegment to hybridize with the antisense strand of the other DNA segment,under appropriate conditions, to form a double helix. A “complement” isdefined as a sequence which pairs to a given sequence based upon thecanonic base-pairing rules. For example, a sequence A-G-T in onenucleotide strand is “complementary” to T-C-A in the other strand.

[0062] In the double helix, adenine appears in one strand, thymineappears in the other strand. Similarly, wherever guanine is found in onestrand, cytosine is found in the other. The greater the relatednessbetween the nucleotide sequences of two DNA segments, the greater theability to form hybrid duplexes between the strands of the two DNAsegments.

[0063] “Similarity” between two amino acid sequences is defined as thepresence of a series of identical as well as conserved amino acidresidues in both sequences. The higher the degree of similarity betweentwo amino acid sequences, the higher the correspondence, sameness orequivalence of the two sequences. (“Identity between two amino acidsequences is defined as the presence of a series of exactly alike orinvariant amino acid residues in both sequences.) The definitions of“complementarity”, “identity” and “similarity” are well known to thoseof ordinary skill in the art.

[0064] “Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 amino acids, morepreferably at least 8 amino acids, and even more preferably at least 15amino acids from a polypeptide encoded by the nucleic acid sequence.

[0065] The present invention also encompasses an isolated nucleotidesequence which encodes PUFA desaturase activity and that ishybridizable, under moderately stringent conditions, to a nucleic acidhaving a nucleotide sequence comprising or complementary to thenucleotide sequence comprising SEQ ID NO:25 or SEQ ID NO:41. A nucleicacid molecule is “hybridizable” to another nucleic acid molecule when asingle-stranded form of the nucleic acid molecule can anneal to theother nucleic acid molecule under the appropriate conditions oftemperature and ionic strength (see Sambrook et al., “Molecular Cloning:A Laboratory Manual, Second Edition (1989), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.)). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. “Hybridization” requires that two nucleic acids containcomplementary sequences. However, depending on the stringency of thehybridization, mismatches between bases may occur. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation. Such variables are wellknown in the art. More specifically, the greater the degree ofsimilarity or homology between two nucleotide sequences, the greater thevalue of Tm for hybrids of nucleic acids having those sequences. Forhybrids of greater than 100 nucleotides in length, equations forcalculating Tm have been derived (see Sambrook et al., supra). Forhybridization with shorter nucleic acids, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra).

[0066] As used herein, an “isolated nucleic acid fragment or sequence”is a polymer of RNA or DNA that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAmay be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA. (A “fragment” of a specified polynucleotide refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10 nucleotides, and evenmore preferably at least about 15 nucleotides, and most preferable atleast about 25 nucleotides identical or complementary to a region of thespecified nucleotide sequence.) Nucleotides (usually found in their5′-monophosphate form) are referred to by their single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

[0067] The terms “fragment or subfragment that is functionallyequivalent” and “functionally equivalent fragment or subfragment” areused interchangeably herein. These terms refer to a portion orsubsequence of an isolated nucleic acid fragment in which the ability toalter gene expression or produce a certain phenotype is retained whetheror not the fragment or subfragment encodes an active enzyme. Forexample, the fragment or subfragment can be used in the design ofchimeric constructs to produce the desired phenotype in a transformedplant. Chimeric constructs can be designed for use in co-suppression orantisense by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the appropriateorientation relative to a plant promoter sequence.

[0068] The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

[0069] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence.

[0070] “Native gene” refers to a gene as found in nature with its ownregulatory sequences. In contrast, “chimeric construct” refers to acombination of nucleic acid fragments that are not normally foundtogether in nature. Accordingly, a chimeric construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than thatnormally found in nature. (The term “isolated” means that the sequenceis removed from its natural environment.)

[0071] A “foreign” gene refers to a gene not normally found in the hostorganism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric constructs. A “transgene” is a genethat has been introduced into the genome by a transformation procedure.

[0072] “Coding sequence” refers to a DNA sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

[0073] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoter sequences canalso be located within the transcribed portions of genes, and/ordownstream of the transcribed sequences. Promoters may be derived intheir entirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (1989) Biochemistry of Plants 15:1-82. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

[0074] An “intron” is an intervening sequence in a gene that does notencode a portion of the protein sequence. Thus, such sequences aretranscribed into RNA but are then excised and are not translated. Theterm is also used for the excised RNA sequences. An “exon” is a portionof the sequence of a gene that is transcribed and is found in the maturemessenger RNA derived from the gene, but is not necessarily a part ofthe sequence that encodes the final gene product.

[0075] The “translation leader sequence” refers to a DNA sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

[0076] The “3” non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., (1989) PlantCell 1:671-680.

[0077] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the Klenow fragment of DNA polymerase I. “Sense” RNA refersto RNA transcript that includes the mRNA and can be translated intoprotein within a cell or in vitro. “Antisense RNA” refers to an RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNAthat may not be translated but yet has an effect on cellular processes.The terms “complement” and “reverse complement” are used interchangeablyherein with respect to mRNA transcripts, and are meant to define theantisense RNA of the message.

[0078] The term “endogenous RNA” refers to any RNA which is encoded byany nucleic acid sequence present in the genome of the host prior totransformation with the recombinant construct of the present invention,whether naturally-occurring or non-naturally occurring, i.e., introducedby recombinant means, mutagenesis, etc.

[0079] The term “non-naturally occurring” means artificial, notconsistent with what is normally found in nature.

[0080] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is regulated by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of regulating theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

[0081] The term “expression”, as used herein, refers to the productionof a functional end-product. Expression of a gene involves transcriptionof the gene and translation of the mRNA into a precursor or matureprotein. “Antisense inhibition” refers to the production of antisenseRNA transcripts capable of suppressing the expression of the targetprotein. “Co-suppression” refers to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020).

[0082] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0083] “Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The preferredmethod of cell transformation of rice, corn and other monocots is theuse of particle-accelerated or “gene gun” transformation technology(Klein et al., (1987) Nature (London) 327:70-73; U.S. Pat. No.4,945,050), or an Agrobacterium-mediated method using an appropriate Tiplasmid containing the transgene (Ishida Y. et al., 1996, NatureBiotech. 14:745-750). The term “transformation” as used herein refers toboth stable transformation and transient transformation.

[0084] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory Press: Cold SpringHarbor, 1989 (hereinafter “Sambrook”).

[0085] The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

[0086] “PCR” or “Polymerase Chain Reaction” is a technique for thesynthesis of large quantities of specific DNA segments, consists of aseries of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk,Conn.). Typically, the double stranded DNA is heat denatured, the twoprimers complementary to the 3′ boundaries of the target segment areannealed at low temperature and then extended at an intermediatetemperature. One set of these three consecutive steps is referred to asa cycle.

[0087] Polymerase chain reaction (“PCR”) is a powerful technique used toamplify DNA millions of fold, by repeated replication of a template, ina short period of time. (Mullis et al, Cold Spring Harbor Symp. Quant.Biol. 51:263-273 (1986); Erlich et al, European Patent Application50,424; European Patent Application 84,796; European Patent Application258,017, European Patent Application 237,362; Mullis, European PatentApplication 201,184, Mullis et al U.S. Pat. No. 4,683,202; Erlich, U.S.Pat. No. 4,582,788; and Saiki et al, U.S. Pat. No. 4,683,194). Theprocess utilizes sets of specific in vitro synthesized oligonucleotidesto prime DNA synthesis. The design of the primers is dependent upon thesequences of DNA that are desired to be analyzed. The technique iscarried out through many cycles (usually 20-50) of melting the templateat high temperature, allowing the primers to anneal to complementarysequences within the template and then replicating the template with DNApolymerase.

[0088] The products of PCR reactions are analyzed by separation inagarose gels followed by ethidium bromide staining and visualizationwith UV transillumination. Alternatively, radioactive dNTPs can be addedto the PCR in order to incorporate label into the products. In this casethe products of PCR are visualized by exposure of the gel to x-ray film.The added advantage of radiolabeling PCR products is that the levels ofindividual amplification products can be quantitated.

[0089] The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell using standard methodology well knownto one skilled in the art. Such construct may be itself or may be usedin conjunction with a vector. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostplants as is well known to those skilled in the art. For example, aplasmid vector can be used. The skilled artisan is well aware of thegenetic elements that must be present on the vector in order tosuccessfully transform, select and propagate host cells comprising anyof the isolated nucleic acid fragments of the invention. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

[0090] Expression of the Omega-3-Desaturase and the Delta 12-DesaturaseGenes

[0091] Once the genes encoding the omega-3 and delta-12 desaturaseenzymes have been isolated, they may then be introduced into either aprokaryotic or eukaryotic host cell (individually or in combination)through the use of a vector or construct. The vector, for example, abacteriophage, cosmid or plasmid, may comprise the nucleotide sequenceencoding either or both of the desaturase enzymes, as well as anypromoter which is functional in the host cell and is able to elicitexpression of the desaturase(s) encoded by the nucleotide sequence(s).The promoter is in operable association with, or operably linked, to thenucleotide sequence. (As noted above, a regulatory sequence (e.g., apromoter) is said to be “operably linked” with a coding sequence if theregulatory sequence affects transcription or expression of the codingsequence. The promoter (or other type of regulatory sequence) need notbe directly linked to the coding sequence. Suitable promoters include,for example, those from genes encoding alcohol dehydrogenase,glyceraldehyde-3 phosphate dehydrogenase, phosphoglucoisomerase,phosphoglycerate kinase, acid phosphatase, T7, TPI, lactase,metallothionein, cytomegalovirus immediate early, whey acidic protein,glucoamylase, and promoters activated in the presence of galactose, forexample, GAL1 and GAL10. Additionally, nucleotide sequences which encodeother proteins, oligosaccharides, lipids, etc. may also be includedwithin the vector as well as other regulatory sequences such as apolyadenylation signal (e.g., the poly-A signal of SV-40T-antigen,ovalalbumin or bovine growth hormone), antibiotic resistance markers,auxotrophic markers, and the like. The choice of sequences present inthe construct is dependent upon the desired expression products, thenature of the host cell, and the proposed means to separate transformedcells from non-transformed cells.

[0092] As noted above, once the vector has been constructed, it may thenbe introduced into the host cell of choice by methods known to those ofordinary skill in the art including, for example, transfection,transformation and electroporation (see Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press (1989)). The host cell is then cultured under suitableconditions permitting expression of the genes leading to the productionof the desired PUFA, which is then recovered and purified. (Thesubstrates which may be produced by the host cell either naturally ortransgenically, as well as the enzymes which may be encoded by DNAsequences present in the vector, which is subsequently introduced intothe host cell, are shown in FIG. 1.)

[0093] Examples of suitable prokaryotic host cells include, for example,bacteria such as Escherichia coli and Bacillus subtilis, as well ascyanobacteria such as Spirulina spp. (i.e., blue-green algae). Examplesof suitable eukaryotic host cells include, for example, mammalian cells,plant cells, yeast cells such as Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Lipomyces starkey, Candida spp. such as Yarrowia(Candida) lipolytica, Kluyveromyces spp., Pichia spp., Trichoderma spp.or Hansenula spp., or fungal cells such as filamentous fungal cells, forexample, Aspergillus, Neurospora and Penicillium. Preferably,Saccharomyces cerevisiae (baker's yeast) cells are utilized.

[0094] Expression in a host cell can be accomplished in a transient orstable fashion. Transient expression can occur from introducedconstructs which contain expression signals functional in the host cell,but which constructs do not replicate and rarely integrate into the hostcell, or where the host cell is not proliferating. Transient expressionalso can be accomplished by inducing the activity of a regulatablepromoter operably linked to the gene of interest, although suchinducible systems frequently exhibit a low basal level of expression.Stable expression can be achieved by introducing a construct that canintegrate into the host genome or that autonomously replicates in thehost cell. Stable expression of the gene of interest can be selectedthrough the use of a selectable marker located on or co-transfected withthe expression construct, followed by selection for cells expressing themarker. When stable expression results from integration, the site of theconstruct's integration can occur randomly within the host genome or canbe targeted through the use of constructs containing regions of homologywith the host genome sufficient to target recombination with the hostlocus. Where constructs are targeted to an endogenous locus, all or someof the transcriptional and translational regulatory regions can beprovided by the endogenous locus.

[0095] A transgenic mammal may also be used in order to express theenzymes of the present invention, and thus ultimately produce thePUFA(s) of interest. More specifically, once the above-describedconstruct is created, it may be inserted into the pronucleus of anembryo. The embryo may then be implanted into a recipient female.Alternatively, a nuclear transfer method can also be utilized (Schniekeet al., Science 278:2130-2133 (1997)). Gestation and birth are thenpermitted to occur (see, e.g., U.S. Pat. No. 5,750,176 and U.S. Pat. No.5,700,671). Milk, tissue, or other fluid samples from the offspringshould then contain altered levels of PUFAs, as compared to the levelsnormally found in the non-transgenic animal. Subsequent generations maybe monitored for production of the altered or enhanced levels of PUFAsand thus incorporation of the gene(s) encoding the desired desaturaseenzyme(s) into their genomes. The mammal utilized as the host may beselected from the group consisting of, for example, mice, rats, rabbits,swine (porcines), goats and sheep (ovines), horses, and bovines.However, any mammal may be used provided it has the ability toincorporate DNA encoding the enzyme of interest into its genome.

[0096] For expression of a desaturase polypeptide, functionaltranscriptional and translational initiation and termination regions areoperably linked to the DNA encoding the desaturase polypeptide ofinterest. Transcriptional and translational initiation and terminationregions are derived from a variety of nonexclusive sources, includingthe DNA to be expressed, genes known or suspected to be capable ofexpression in the desired system, expression vectors, chemicalsynthesis, or from an endogenous locus in a host cell. Expression in aplant tissue and/or plant part presents certain efficiencies,particularly where the tissue or part is one which is harvested early,such as seed, leaves, fruits, flowers, roots, etc. Expression can betargeted to that location with the plant by utilizing specificregulatory sequence such as those of U.S. Pat. Nos. 5,463,174;4,943,674; 5,106,739; 5,175,095; 5,420,034; 5,188,958; and 5,589,379.

[0097] Alternatively, the expressed protein can be an enzyme thatproduces a product, and that product may be incorporated, eitherdirectly or upon further modifications, into a fluid fraction from thehost plant. Expression of a desaturase gene, or antisense desaturasetranscripts, can alter the levels of specific PUFAs, or derivativesthereof, found in plant parts and/or plant tissues. The desaturasepolypeptide coding region may be expressed either by itself or withother genes, in order to produce tissues and/or plant parts containinghigher proportions of desired PUFAs, or in which the PUFA compositionmore closely resembles that of human breast milk (Prieto et al., PCTpublication WO 95/24494).

[0098] The termination region may be derived from the 3′ region of thegene from which the initiation region was obtained or from a differentgene. A large number of termination regions are known to be satisfactoryin a variety of hosts from the same and different genera and species.The termination region usually is selected as a matter of conveniencerather than because of any particular property.

[0099] As noted above, a plant (e.g., Glycine max (soybean) or Brassicanapus (canola)) or plant tissue may also be utilized as a host or hostcell, respectively, for expression of the desaturase enzymes, which may,in turn, be utilized in the production of PUFAs. More specifically,desired PUFAS can be expressed in seed. Methods of isolating seed oilsare known in the art. Thus, in addition to providing a source for PUFAs,seed oil components may be manipulated through the expression of thedesaturase genes, as well as perhaps other desaturase genes and elongasegenes, to provide seed oils that can be added to nutritionalcompositions, pharmaceutical compositions, animal feeds and cosmetics.Once again, a vector that comprises a DNA sequence encoding thedesaturase gene of interest, operably linked to a promoter, isintroduced into the plant tissue or plant for a time and underconditions sufficient for expression of the desaturase gene. The vectormay also comprise one or more genes that encode other enzymes, forexample, delta-5-desaturase, elongase, delta-12-desaturase,delta-15-desaturase, delta-17-desaturase, and/or delta-19-desaturaseenzymes. The plant tissue or plant may produce the relevant substrate(e.g., adrenic acid or DPA) upon which the enzyme acts or a vectorencoding enzymes that produce such substrates may be introduced into theplant tissue, plant cell or plant. In addition, suitable substrates maybe sprayed on plant tissues expressing the appropriate enzymes. Usingthese various techniques, one may produce PUFAs by use of a plant cell,plant tissue, or plant. It should also be noted that the invention alsoencompasses a transgenic plant comprising the above-described vector,wherein expression of the nucleotide sequence(s) of the vector resultsin production of a desired PUFA in, for example, the seeds of thetransgenic plant.

[0100] The regeneration, development, and cultivation of plants fromsingle plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In: Methodsfor Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego,Calif., (1988)). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

[0101] The development or regeneration of plants containing the foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

[0102] There are a variety of methods for the regeneration of plantsfrom plant tissue. The particular method of regeneration will depend onthe starting plant tissue and the particular plant species to beregenerated.

[0103] Methods for transforming dicots, primarily by use ofAgrobacterium tumefaciens, and obtaining transgenic plants have beenpublished for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135,U.S. Pat. No. 5,518, 908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat.No. 5,416,011, McCabe et. al., BiolTechnology 6:923 (1988), Christou etal., Plant Physiol. 87:671-674 (1988)); Brassica (U.S. Pat. No.5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653-657 (1996),McKently et al., Plant Cell Rep. 14:699-703 (1995)); papaya; and pea(Grant et al., Plant Cell Rep. 15:254-258, (1995)).

[0104] Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204(1988), Gordon-Kamm et al., Plant Cell 2:603-618 (1990), Fromm et al.,BiolTechnology 8:833 (1990), Koziel et al., BiolTechnology 11: 194,(1993), Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., BiolTechnology 10: 15 89 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet.205:34, (1986); Part et al., Plant Mol. Biol. 32:1135-1148, (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep.7:379, (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., BiolTechnology 10:691 (1992)), and wheat(Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

[0105] Assays for gene expression based on the transient expression ofcloned nucleic acid constructs have been developed by introducing thenucleic acid molecules into plant cells by polyethylene glycoltreatment, electroporation, or particle bombardment (Marcotte et al.,Nature 335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)).

[0106] Transient expression systems may be used to functionally dissectgene constructs (see generally, Maliga et al., Methods in PlantMolecular Biology, Cold Spring Harbor Press (1995)). It is understoodthat any of the nucleic acid molecules of the present invention can beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promoters,enhancers etc.

[0107] In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, N.Y. (1998); Plant Molecular Biology: A LaboratoryManual, eds. Clark, Springer, New York (1997)).

[0108] In view of the above, the present invention encompasses a methodof producing an omega-3 desaturase and/or a delta-12 desaturase enzyme,the method comprising the steps of: 1) isolating the nucleotide sequenceof the gene encoding the desired desaturase enzyme(s); 2) constructing avector comprising the nucleotide sequence(s); and 3) introducing thevector into a host cell for a time and under conditions sufficient forthe production of the desaturase enzyme(s).

[0109] The present invention also encompasses a method of producingPUFAs, the method comprising exposing a suitable fatty acid substrate tothe enzyme such that the desaturase converts the fatty acid substrate toa desired PUFA product. For example, when AA (20:4n-6) is exposed to theomega-3 desaturase enzyme of the present invention, it is converted intoEPA (20:5n-3). The EPA so formed may be converted into DPA (22:5n-3) bythe action of an elongase, and the DPA subsequently converted into DHA(22:6n-3) by a delta-4 desaturase.

[0110] Likewise, when OA (18:1n-9) is exposed to the delta-12 desaturaseenzyme of the present invention, it is converted into LA (18:2n-6). TheLA so formed may be converted into virtually all of the PUFAs shown inFIG. 1 by the subsequent actions of suitable desaturases and/orelongases.

[0111] Uses of the Subject Desaturase Genes and Enzymes Encoded Thereby:

[0112] As noted above, the isolated desaturase genes and the desaturaseenzymes encoded thereby have many uses. For example, the genes and thecorresponding enzymes may be used indirectly or directly, singly or incombination, in the production of PUFAs. For example, the omega-3desaturase may be used in the production of ETA, EPA, DPA, DHA, and thelike. As used in this context, the word “directly” encompasses thesituation where the enzyme is used to catalyze the conversion of a fattyacid substrate directly into the desired fatty acid product, without anyintermediate steps or pathway intermediates (e.g., the conversion of AAto EPA). The product so obtained is then utilized in a composition.“Indirectly” encompasses the situation where a desaturase according tothe present invention is used to catalyze the conversion of a fatty acidsubstrate into another fatty acid (i.e., a pathway intermediate) by thedesaturase (e.g., the conversion of AA to EPA) and then the latter fattyacid (the EPA) is converted to the desired fatty acid product by use ofanother desaturase or non-desaturase enzyme (e.g., the conversion of EPAto DPA by elongase). These PUFAs (i.e., those produced either directlyor indirectly by the activity of the subject desaturases) may be addedto, for example, nutritional compositions, pharmaceutical compositions,cosmetics, and animal feeds, all of which are encompassed by the presentinvention. Such uses are described in detail below.

[0113] Nutritional Compositions:

[0114] The present invention includes nutritional compositions. Forpurposes of the present invention, such compositions include any food orpreparation for human consumption (including for enteral and/orparenteral consumption) which when taken into the body (a) serve tonourish or build up tissues or supply energy; and/or (b) maintain,restore or support adequate nutritional status or metabolic function.

[0115] The nutritional composition of the present invention comprises anoil, fatty acid ester, or fatty acid produced directly or indirectly byuse of the desaturase genes disclosed herein. The composition may eitherbe in a solid or liquid form. Additionally, the composition may includeedible macronutrients, vitamins, and/or minerals in amounts desired fora particular use. The amounts of these ingredients will vary dependingon whether the composition is intended for use with normal, healthyinfants, children, or adults, or for use with individuals havingspecialized needs, such as individuals suffering from metabolicdisorders and the like.

[0116] Examples of macronutrients that may be added to the compositionsinclude (but are not limited to): edible fats, carbohydrates andproteins. Examples of such edible fats include (but are not limited to):coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and diglycerides. Examples of such carbohydrates include (but arenot limited to): glucose, edible lactose, and hydrolyzed search.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include (but are not limitedto) soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

[0117] With respect to vitamins and minerals, the following may be addedto the nutritional compositions of the present invention: calcium,phosphorus, potassium, sodium, chloride, magnesium, manganese, iron,copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the Bcomplex. Other such vitamins and minerals may also be added.

[0118] The components utilized in the nutritional compositions of thepresent invention will be of semi-purified or purified origin. Bysemi-purified or purified is meant a material which has been prepared bypurification of a natural material or by de novo synthesis.

[0119] Examples of nutritional compositions of the present inventioninclude (but are not limited to): infant formulas, dietary supplements,dietary substitutes, and rehydration compositions. Nutritionalcompositions of particular interest include (but are not limited to)compositions for enteral and parenteral supplementation for infants,specialized infant formulas, supplements for the elderly, andsupplements for those with gastrointestinal difficulties and/ormalabsorption.

[0120] The nutritional composition of the present invention may also beadded to food even when supplementation of the diet is not required. Forexample, the composition may be added to food of any type, including(but not limited to): margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

[0121] In a preferred embodiment of the present invention, thenutritional composition is an enteral nutritional product, morepreferably, an adult or pediatric enteral nutritional product. Thiscomposition may be administered to adults or children experiencingstress or having specialized needs due to chronic or acute diseasestates. The composition may comprise, in addition to PUFAs producedaccording to the present invention, macronutrients, vitamins, and/orminerals, as described previously. The macronutrients may be present inamounts equivalent to those present in human milk or on an energy basis,i.e., on a per calorie basis.

[0122] The enteral formula, for example, may be sterilized andsubsequently utilized on a ready-to-feed (RTF) basis or stored in aconcentrated liquid or powder. The powder can be prepared byspray-drying the formula prepared as indicated above, and reconstitutingit by rehydrating the concentrate. Adult and pediatric nutritionalformulas are known in the art and are commercially available (e.g.,Similac®, Ensure@, Jevity® and Alimentum® from Ross Products Division,Abbott Laboratories, Columbus, Ohio). An oil or fatty acid produced inaccordance with the present invention may be added to any of theseformulas.

[0123] The energy density of the nutritional compositions of the presentinvention, when in liquid form, may range from about 0.6 kcal to about 3kcal per ml. When in solid or powdered form, the nutritional supplementsmay contain from about 1.2 to more than 9 kcals per gram, preferablyabout 3 to 7 kcals per gm. In general, the osmolality of a liquidproduct should be less than 700 mOsm and, more preferably, less than 660mOsm.

[0124] The nutritional formula may include macronutrients, vitamins, andminerals, as noted above, in addition to the PUFAs produced inaccordance with the present invention. The presence of these additionalcomponents helps the individual ingest the minimum daily requirements ofthese elements. In addition to the provision of PUFAs, it may also bedesirable to add zinc, copper, folic acid, and antioxidants to thecomposition. It is believed that these substances boost a stressedimmune system and will therefore provide further benefits to theindividual receiving the composition. A pharmaceutical composition mayalso be supplemented with these elements.

[0125] In a more preferred embodiment, the nutritional compositioncomprises, in addition to antioxidants and at least one PUFA, a sourceof carbohydrate wherein at least 5 weight percent of the carbohydrate isindigestible oligosaccharide. In a more preferred embodiment, thenutritional composition additionally comprises protein, taurine, andcarnitine.

[0126] As noted above, the PUFAs produced in accordance with the presentinvention, or derivatives thereof, may be added to a dietary substituteor supplement, particularly an infant formula, for patients undergoingintravenous feeding or for preventing or treating malnutrition or otherconditions or disease states. As background, it should be noted thathuman breast milk has a fatty acid profile comprising from about 0.15%to about 0.36% DHA, from about 0.03% to about 0.13% EPA, from about0.30% to about 0.88% AA, from about 0.22% to about 0.67% DGLA, and fromabout 0.27% to about 1.04% GLA. Thus, fatty acids such as AA, EPA and/orDHA produced in accordance with the present invention can be used toalter, for example, the composition of infant formulas in order toreplicate more faithfully the PUFA content of human breast milk or toalter the presence of PUFAs normally found in a non-human mammal's milk.In particular, a composition for use a medicinal agent or foodsupplement, particularly a breast milk substitute or supplement, willpreferably comprise one or more of AA, DGLA and GLA. More preferably,the composition will comprise from about 0.3 to 30% AA, from about 0.2to 30% DGLA, and/or from about 0.2 to about 30% GLA.

[0127] Parenteral nutritional compositions comprising from about 2 toabout 30% by weight fatty acids calculated as triglycerides areencompassed by the present invention. The preferred composition hasabout 1 to about 25% by weight of the total PUFA composition as GLA.Other vitamins, particularly fat-soluble vitamins such as vitamin A, D,E and L-carnitine, can optionally be included. When desired, apreservative such as alpha-tocopherol may be added in an amount of about0.1% by weight.

[0128] In addition, the ratios of AA, DGLA and GLA can be adapted for aparticular given end use. When formulated as a breast milk supplement orsubstitute, a composition which comprises one or more of AA, DGLA andGLA will be provided in a ratio of from about 1:19:30 to about 6:1:0.2,respectively. For example, the breast milk of animals can vary in ratiosof AA:DGLA:GLA ranging from 1:19:30 to 6:1:0.2, which includesintermediate ratios which are preferably about 1:1:1, 1:2:1, 1:1:4. Whenproduced together in a host cell, adjusting the rate and percent ofconversion of a precursor substrate such as GLA and DGLA to AA can beused to control the PUFA ratios precisely. For example, a 5% to 10%conversion rate of DGLA to AA can be used to produce an AA to DGLA ratioof about 1:19, whereas a conversion rate of about 75% to 80% can be usedto produce an AA to DGLA ratio of about 6:1. Therefore, whether in acell culture system or in a host animal, regulating the timing, extentand specificity of desaturase expression, as well as the expression ofother desaturases and elongases, can be used to modulate PUFA levels andratios. The PUFAs produced in accordance with the present invention(e.g., AA, EPA, etc.) may then be combined with other PUFAs or othertypes of fatty acids in the desired concentrations and ratios.

[0129] Additionally, PUFAs produced in accordance with the presentinvention or host cells transformed to contain and express the subjectdesaturase genes may also be used as animal food supplements to alter ananimal's tissue or milk fatty acid composition to one more desirable forhuman or animal consumption.

[0130] Pharmaceutical Compositions:

[0131] The present invention also encompasses a pharmaceuticalcomposition comprising one or more of the acids and/or resulting oilsproduced using the desaturase genes, in accordance with the methodsdescribed herein. Specifically, such a pharmaceutical composition maycomprise one or more of the PUFAs and/or oils, in combination with astandard, well-known, non-toxic pharmaceutically-acceptable carrier,adjuvant or vehicle such as phosphate-buffered saline, water, ethanol,polyols, vegetable oils, a wetting agent or an emulsion such as awater/oil emulsion. The composition may be in either a liquid or solidform. For example, the composition may be in the form of a tablet,capsule, ingestible liquid or powder, injectible, or topical ointment orcream. Proper fluidity can be maintained, for example, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. It may also be desirable to include isotonicagents, for example, sugars, sodium chloride, and the like. Besides suchinert diluents, the composition can also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening agents,flavoring agents and perfuming agents.

[0132] Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanthor mixtures of these substances.

[0133] Solid dosage forms such as tablets and capsules can be preparedusing techniques well known in the art. For example, PUFAs produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant PUFA(s). The antioxidant and PUFAcomponents should fit within the guidelines presented above.

[0134] For intravenous administration, the PUFAs produced in accordancewith the present invention or derivatives thereof may be incorporatedinto commercial formulations such as Intralipids™. The typical normaladult plasma fatty acid profile comprises 6.64 to 9.46% AA, 1.45 to3.11% DGLA, and 0.02 to 0.08% GLA. These PUFAs or their metabolicprecursors can be administered alone or in combination with other PUFAsto achieve a normal fatty acid profile in a patient. Where desired, theindividual components of the formulations may be provided individually,or in kit form, for single or multiple use. A typical dosage of aparticular fatty acid is from 0.1 mg to 20 g, taken from one to fivetimes per day (up to 100 g daily) and is preferably in the range of fromabout 10 mg to about 1, 2, 5, or 10 g daily (taken in one or multipledoses).

[0135] Possible routes of administration of the pharmaceuticalcompositions of the present invention include, for example, enteral(e.g., oral and rectal) and parenteral. For example, a liquidpreparation may be administered orally or rectally. Additionally, ahomogenous mixture can be completely dispersed in water, admixed understerile conditions with physiologically acceptable diluents,preservatives, buffers or propellants to form a spray or inhalant.

[0136] The route of administration will, of course, depend upon thedesired effect. For example, if the composition is being utilized totreat rough, dry, or aging skin, to treat injured or burned skin, or totreat skin or hair affected by a disease or condition, it may be appliedtopically.

[0137] The dosage of the composition to be administered to the patientmay be determined by one of ordinary skill in the art and depends uponvarious factors such as weight of the patient, age of the patient,overall health of the patient, past history of the patient, immunestatus of the patient, etc.

[0138] With respect to form, the composition may be, for example, asolution, a dispersion, a suspension, an emulsion or a sterile powderthat is then reconstituted.

[0139] The present invention also includes the treatment of variousdisorders by use of the pharmaceutical and/or nutritional compositionsdescribed herein. In particular, the compositions of the presentinvention may be used to treat restenosis after angioplasty.Furthermore, symptoms of inflammation, rheumatoid arthritis, asthma andpsoriasis may also be treated with the compositions of the invention.Evidence also indicates that PUFAs may be involved in calciummetabolism; thus, the compositions of the present invention may beutilized in the treatment or prevention of osteoporosis and of kidney orurinary tract stones.

[0140] Additionally, the compositions of the present invention may alsobe used in the treatment of cancer. Malignant cells have been shown tohave altered fatty acid compositions. Addition of fatty acids has beenshown to slow their growth, cause cell death and increase theirsusceptibility to chemotherapeutic agents. Moreover, the compositions ofthe present invention may also be useful for treating cachexiaassociated with cancer.

[0141] The compositions of the present invention may also be used totreat diabetes (see U.S. Pat. No. 4,826,877 and Horrobin et al., Am. J.Clin. Nutr. Vol. 57 (Suppl.) 732S-737S). Altered fatty acid metabolismand composition have been demonstrated in diabetic animals.

[0142] Furthermore, the compositions of the present invention,comprising PUFAs produced either directly or indirectly through the useof the desaturase enzymes, may also be used in the treatment of eczemaand in the reduction of blood pressure. Additionally, the compositionsof the present invention may be used to inhibit platelet aggregation, toinduce vasodilation, to reduce cholesterol levels, to inhibitproliferation of vessel wall smooth muscle and fibrous tissue (Brenneret al., Adv. Exp. Med. Biol. Vol. 83, p.85-101, 1976), to reduce or toprevent gastrointestinal bleeding and other side effects ofnon-steroidal anti-inflammatory drugs (see U.S. Pat. No. 4,666,701), toprevent or to treat endometriosis and premenstrual syndrome (see U.S.Pat. No. 4,758,592), and to treat myalgic encephalomyelitis and chronicfatigue after viral infections (see U.S. Pat. No. 5,116,871).

[0143] Further uses of the compositions of the present invention includeuse in the treatment of AIDS, multiple sclerosis, and inflammatory skindisorders, as well as for maintenance of general health.

[0144] Additionally, the composition of the present invention may beutilized for cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or a PUFA produced accordingto the subject invention may be used as the sole “active” ingredient ina cosmetic composition.

[0145] Veterinary Applications:

[0146] It should be noted that the above-described pharmaceutical andnutritional compositions may be utilized in connection with animals(i.e., domestic or non-domestic, including mammals, birds, reptiles,lizards, etc.), as well as humans, as animals experience many of thesame needs and conditions as humans. For example, the oil or fatty acidsof the present invention may be utilized in animal feed supplements,animal feed substitutes, animal vitamins or in animal topical ointments.

[0147] The present invention may be further illustrated by thenon-limiting examples presented below:

EXAMPLE 1 Construction of Saprolegnia diclina (ATCC 56851)cDNA Library

[0148] To isolate genes encoding for functional desaturase enzymes, acDNA library was constructed. Saprolegnia diclina cultures were grown inpotato dextrose media (Difco # 336, BD Diagnostic Systems, Sparks,Maryland) at room temperature for four days with constant agitation. Themycelia were harvested by filtration through several layers ofcheesecloth, and the cultures were crushed in liquid nitrogen using amortar and pestle. The cell lysates were resuspended in RT buffer(Qiagen, Valencia, Calif.) containing β-mercaptoethanol and incubated at55° C. for three minutes. These lysates were homogenized either byrepeated aspirations through a syringe or over a “Qiashredder”-brandcolumn (Qiagen). The total RNA was finally purified using the “RNeasyMaxi”-brand kit (Qiagen), as per the manufacturer's protocol.

[0149] mRNA was isolated from total RNA from each organism using anoligo dT cellulose resin. The “pBluescript II XR”-brand libraryconstruction kit (Stratagene, La Jolla, Calif.) was used to synthesizedouble-stranded cDNA. The double-stranded cDNA was then directionallycloned (5′ EcoRI/3′ XhoI) into pBluescript II SK(+) vector (Stratagene).The S. diclina library contained approximately 2.5×10⁶ clones, each withan average insert size of approximately 700 bp. Genomic DNA of S.diclina was isolated by crushing the culture in liquid nitrogen followedby purification using the “Genomic DNA Extraction”-brand kit (Qiagen),as per the manufacturer's protocol.

EXAMPLE 2 Determination of Codon Usage in Saprolegnia diclina

[0150] The 5′ ends of 350 random cDNA clones were sequenced from theSaprolegnia diclina cDNA library described in Example 1. The sequenceswere translated into six reading frames using GCG program (GeneticsComputer Group, Madison, Wis.) with the “FastA”-brand algorithm tosearch for similarity between a query sequence and a group of sequencesof the same type, specifically within the GenBank database. Many of theclones were identified as putative housekeeping genes based on proteinhomology to known genes. Eight S. diclina cDNA sequences were thusselected. Additionally, the full-length S. diclina delta 5-desaturaseand delta 6-desaturase sequences were also used (see Table 1 below).These sequences were then used to generate the S. diclina codon biastable shown in Table 2 below by employing the “CodonFrequency” programfrom GCG. TABLE 1 Genes from Saprolegnia diclina used for generation ofCodon Bias Table # amino Clone # Match # bases acids 3 Actin gene 615205 20 Ribosomal protein L23 420 140 55 Heat Shock protein 70 gene 468156 83 Glyceraldehyde-3-P-dehydrogenase 588 196 gene 138 Ribosomalprotein S13 gene 329 110 179 Alpha-tubulin 3 gene 591 197 190 Caseinkinase II alpha subunit 627 209 gene 250 Cyclophilin gene 489 163 Delta6-desaturase 1362 453 Delta 5-desaturase 1413 471 Total 6573 2191

[0151] TABLE 2 Codon Bias Table for Saprolegnia diclina Amino acid CodonBias % used Ala GCC 55% Arg CGC 50% Asn AAC 94% Asp GAC 85% Cys TGC 77%Gln CAG 90% Glu GAG 80% Gly GGC 67% His CAC 86% Ile ATC 82% Leu CTC 52%Lys AAG 87% Met ATG 100% Phe TTC 72% Pro CCG 55% Ser TCG 47% Thr ACG 46%Trp TGG 100% Tyr TAC 90% Val GTC 73% Stop TGA 67%

EXAMPLE 3 Design of Degenerate Oligonucleotides for the Isolation of anOmega-3 Desaturase from Saprolegnia diclina (ATCC 56851)

[0152] Fungi like Saprolegnia diclina produce a wide range of PUFAs,including arachidonic acid (AA) and eicosapentaenoic acid (EPA) via thePUFA biosynthetic pathway depicted in FIG. 1. Analysis of the fatty acidcomposition of Saprolegnia diclina (ATCC 56851) showed 15.42% of thetotal lipid to be AA and 12.2% of the total lipid to be EPA (see Table5). Linoleic acid (LA) was the only other intermediate present in highamounts. This indicates that S. diclina has very active delta-6 anddelta-5 desaturases, as well as elongases that shunt intermediatesthrough the pathway depicted in FIG. 1. Due to the high percentage ofEPA in this organism, an active omega-3 desaturase (synonymous with a“delta-15” desaturase when the substrate is a C₁₈ fatty acid, a“delta-17” desaturase when the substrate is a C₂₀ fatty acid, and a“delta-19” desaturase when the substrate is a C₂₂ fatty acid) ispredicted to exist which is capable of converting AA (20:4n-6) to EPA(20:5n-3).

[0153] As just noted, omega-3 desaturases are enzymes that catalyze theintroduction of a double bond at the delta-15 position for C₁₈-acylchains, the delta-17 position for C₂₀-acyl chains, and the delta-19position for C₂₂-acyl chains. There are several known omega-3desaturases from plants, but these act exclusively on C₁₈ fatty acidsubstrates like LA (18:2n-6) and GLA (18:3n-6). These types ofdesaturases are collectively referred to as delta 15-desaturases. Atthis point in time, only one omega-3 desaturase gene has been isolatedwhose encoded enzyme catalyzes the desaturation of C₁₈, C₂₀, and C₂₂fatty acid substrates. This is fat-1 from C. elegans. See U.S. Pat. No.6,194,167, issued Feb. 27, 2001.

[0154] The approach used in this Example to identify an omega-3desaturase from S. diclina involved PCR amplification of a region of thedesaturase gene using degenerate oligonucleotides (primers) thatcontained conserved motifs present in other known omega-3 desaturases.Omega-3 desaturases from the following organisms were used for thedesign of these degenerate primers: Arabidopsis thaliana (Swissprot#P46310), Ricunus communis (Swissprot #P48619), Glycine max (Swissprot#P48621), Sesamum indicum (Swissprot #P48620), Nicotiana tabacum(GenBank #D79979), Perilla frutescens (GenBank #U59477), Capsicum annuum(GenBank #AF222989), Limnanthes douglassi (GenBank #U17063), andCaenorhabditis elegans (GenBank #L41807). Some primers were designed tocontain the conserved histidine-box motifs that are important componentsof the active site of the enzymes. See Shanklin, J. E., McDonough, V.M., and Martin, C. E. (1994) Biochemistry 33, 12787-12794.

[0155] Alignment of sequences was carried out using the CLUSTALWMultiple Sequence Alignment Program (http://workbench.sdsc.edu).

[0156] The following degenerate primers were designed and used invarious combinations: Protein Motif 1: NH₃— TRAAIPKHCWVK —COOH Primer RO1144 (Forward): 5′-ATC CGC GCC GCC ATC CCC AAG CAC (SEQ ID NO: 1) TGCTGG GTC AAG-3′.

[0157] Protein Motif 2: NH₃-ALFVLGHDCGHGSFS —COOH

[0158] This primer contains the histidine-box 1 (underlined). Primer RO1119 (Forward): 5′-GCC CTC TTC GTC CTC GGC CAY (SEQ. ID. NO: 2) GAC TGCGGC CAY GGC TCG TTC TCG-3′. Primer RO 1118 (Reverse): 5′-GAG RTG GTA RTGGGG GAT CTG GGG (SEQ. ID. NO: 3) GAA GAR RTG RTG GRY GAC RTG-3′.

[0159] Protein Motif 3: NH₃-PYHGWRISHRTHHQN —COOH

[0160] This primer contains the histidine-box 2 (underlined). Primer RO1121 (Forward): 5′-CCC TAC CAY GGC TGG CGC ATC TCG (SEQ. ID. NO: 4) CAYCGC ACC CAY CAY CAG AAC-3′. Primer RO 1122 (Reverse): 5′-GTT CTG RTG RTGGGT CCG RTG CGA (SEQ. ID. NO: 5) `GAT GCG CCA GCC RTG GTA GGG-3′.Protein Motif 4: NH₃— GSHF D/H P D/Y SDLFV —COOH Primer RO 1146(Forward): 5′-GGC TCG CAC TTC SAC CCC KAC (SEQ. ID. NO: 6) TCG GAC CTCTTC GTC-3′. Primer RO 1147 (Reverse): 5′-GAC GAA GAG GTC CGA GTM GGG(SEQ. ID. NO: 7) GTW GAA GTG CGA GCC-3′. Protein Motif 5: NH₃— WS Y/FL/V RGGLTT L/I DR —COOH Primer RO 1148 (Reverse): 5′-GCG CTG GAK GGT GGTGAG GCC (SEQ. ID. NO: 8) GCC GCG GAW GSA CGA CCA-3′.

[0161] Protein Motif 6: NH₃-HHDIGTHVIHHLFPQ —COOH

[0162] This sequence contains the third histidine-box (underlined).Primer RO 1114 (Reverse): 5′-CTG GGG GAA GAG RTG RTG GAT (SEQ. ID. NO:9) GAC RTG GGT GCC GAT GTC RTG RTG-3′. Protein Motif 7: NH₃— H L/F FPQ/K IPHYHL V/I EAT —COOH Primer RO 1116 (Reverse): 5′-GGT GGC CTC GAYGAG RTG GTA (SEQ. ID. NO: 10) RTG GGG GAT CTK GGG GAA GAR RTG-3′.

[0163] Protein Motif 8: NH₃—HV A/I HH L/F FPQIPHYHL —COOH

[0164] This primer contains the third histidine-box (underlined) andaccounts for differences between the plant omege-3 desaturases and theC. elegans omega-3-desaturase. Primer RO 1118 (Reverse): 5′-GAG RTG GTARTG GGG GAT CTG GGG (SEQ. ID. NO: 11) GAA GAR RTG RTG GRY GAC RTG-3′.

[0165] The degeneracy code used for SEQ. ID. NOS: 1 through 11 was asfollows: R=A/G; Y=C/T; M=A/C; K=G/T; W=A/T; S=C/G; B=C/G/T; D=A/G/T;H=A/C/T; V=A/C/G; and N=A/C/G/T.

EXAMPLE 4 Identification and Isolation of an Omega-3 Desaturase Genefrom Saprolegnia diclina (ATCC 56851)

[0166] Various sets of the degenerate primers disclosed in Example 3were used in PCR amplification reactions, using as a template either theS. diclina cDNA library plasmid DNA (from Example 1), or S. diclinagenomic DNA. Also various different DNA polymerases and reactionconditions were used for the PCR amplifications. These reactionsvariously involved using “Platinum Taq”-brand DNA polymerase (LifeTechnologies Inc., Rockville, Md.), or cDNA polymerase (Clonetech, PaloAlto, Calif.), or Taq PCR-mix (Qiagen), at differing annealingtemperatures.

[0167] PCR amplification using the primers RO 1121 (Forward) (SEQ. ID.NO: 4) and RO 1116 (Reverse) (SEQ. ID. NO: 10) resulted in thesuccessful amplification of a fragment homologous to a known omega-3desaturase. The RO 1121 (Forward) primer corresponds to the proteinmotif 3; the RO 1116 (Reverse) primer corresponds to the protein motif7.

[0168] PCR amplification was carried out in a 50 μl total volumecontaining: 3 μl of the cDNA library template, PCR buffer containing 40mM Tricine-KOH (pH 9.2), 15 mM KOAc, 3.5 mM Mg(OAc)₂, 3.75 μg/ml BSA(final concentration), 200 μM each deoxyribonucleotide triphosphate, 10pmole of each primer and 0.5 μl of “Advantage”-brand cDNA polymerase(Clonetech). Amplification was carried out as follows: initialdenaturation at 94° C. for 3 minutes, followed by 35 cycles of thefollowing: 94° C. for 1 min, 60° C. for 30 sec, 72° C. for 1 min. Afinal extension cycle of 72° C. for 7 min was carried out, followed byreaction termination at 4° C.

[0169] A single ˜480 bp PCR band was generated which was resolved on a“SeaKem Gold”-brand agarose gel (FMC BioProducts, Rockland, Me.), andgel-purified using the Qiagen Gel Extraction Kit. The staggered ends onthe fragment were “filled-in” using T4 DNA polymerase (LifeTechnologies, Rockville, Md.) as per the manufacturer's instructions,and the DNA fragments were cloned into the PCR-Blunt vector (Invitrogen,Carlsbad, Calif.). The recombinant plasmids were transformed into TOP10supercompetent cells (Invitrogen), and eight clones were sequenced and adatabase search (Gen-EMBL) was carried out. 50

[0170] Clones “sdd17-7-1” to “sdd17-7-8’ were all found to contain and˜483 bp insert. The deduced amino acid sequence from this fragmentshowed highest identity to the following proteins (based on a “tFastA”search):

[0171] 1. 37.9% identity in 161 amino acid overlap with an omega-3(delta-15) desaturase from Synechocystis sp. (Accession #D13780).

[0172] 2. 40.7% identity in 113 amino acid overlap with Picea abiesplastidial omega-3 desaturase (Accession #AJ302017).

[0173] 3. 35.9% identity in 128 amino acid overlap with Zea mays FAD8fatty acid desaturase (Accession #D63953).

[0174] Based on its sequence homology to known omega-3 fatty aciddesaturases, it seemed likely that this DNA fragment was part of anomega-3 desaturase gene present in S. diclina.

[0175] The DNA sequence identified above was used in the designoligonucleotides to isolate the 3′ and the 5′ ends of this gene from thecDNA library described in Example 1. To isolate the 3′ end of the gene,the following oligonucleotides were designed: RO 1188 (Forward): 5′-TACGCG TAC CTC ACG TAC TCG CTC G-3′. (SEQ. ID. NO: 12) RO 1189 (Forward):5′-TTC TTG CAC CAC AAC GAC GAA GCG ACG-3′. (SEQ. ID. NO: 13) RO 1190(Forward): 5′-GGA GTG GAC GTA CGT CAA GGG CAA C-3′. (SEQ. ID. NO: 14) RO1191 (Forward): 5′-TCA AGG GCA ACC TCT CGA GCG TCG AC-3′. (SEQ. ID. NO:15)

[0176] These primers (SEQ. ID. NOS: 12-15) were used in combinationswith the pBluescript SK(+) vector oligonucleotide: RO 898: 5′-CCC AGTCAC GAC GTT GTA AAA CGA CGG CCA G-3′ (SEQ. ID. NO: 16).

[0177] PCR amplifications were carried out using either the “Taq PCRMaster Mix” brand polymerase (Qiagen) or “Advantage”-brand cDNApolymerase (Clonetech) or “Platinum”-brand Taq DNA polymerase (LifeTechnologies), as follows:

[0178] For the “Taq PCR Master Mix” polymerase, 10 pmoles of each primerwere used along with 1 μl of the cDNA library DNA from Example 1.Amplification was carried out as follows: initial denaturation at 94° C.for 3 min, followed by 35 cycles of the following: 94° C. for 1 min, 60°C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for 7min was carried out, followed by the reaction termination at 4° C. Thisamplification resulted in the most distinct bands as compared with theother two conditions tested.

[0179] For the “Advantage”-brand cDNA polymerase reaction, PCRamplification was carried out in a 50 μl total volume containing: 1 μlof the cDNA library template from Example 1, PCR buffer containing 40 mMTricine-KOH (pH 9.2), 15 mM KOAc, 3.5 mM Mg(OAc)₂, 3.75 μg/ml BSA (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer and 0.5 μl of cDNA polymerase (Clonetech). Amplificationwas carried out as described for the Taq PCR Master Mix.

[0180] For the “Platinum”-brand Taq DNA polymerase reaction, PCRamplification was carried out in a 50 μl total volume containing: 1 μlof the cDNA library template from Example 1, PCR buffer containing 20 mMTris-Cl, pH 8.4, 50 mM KCl (final concentration), 200 μM eachdeoxyribonucleotide triphosphate, 10 pmole of each primer, 1.5 mM MgSO₄,and 0.5 μl of Platinum Taq DNA polymerase. Amplification was carried outas follows: initial denaturation at 94° C. for 3 min, followed by 30cycles of the following: 94° C. for 45 sec, 55° C. for 30 sec, 68° C.for 2 min. The reaction was terminated at 4° C.

[0181] All four sets of primers in combination with the vector primergenerated distinct bands. PCR bands from the combination (RO 1188+RO898) were >500 bp and this was gel-purified and cloned separately. ThePCR bands generated from the other primer combinations were <500 bp. Thebands were gel-purified, pooled together, and cloned into PCR-Bluntvector (Invitrogen) as described earlier. The recombinant plasmids weretransformed into TOP10 supercompetent cells (Invitrogen) and clones weresequenced and analyzed.

[0182] Clone “sdd17-16-4” and “sdd16-6” containing the ˜500 bp insert,and clones “sdd17-17-6,” “sdd17-17-10,” and “sdd17-20-3,” containing the˜400 bp inserts, were all found to contain the 3′-end of the putativeomega-3 desaturase. These sequences overlapped with each other, as wellas with the originally identified fragment of this putative omega-3desaturase gene. All of the sequences contained the ‘TAA’ stop codon anda poly-A tail typical of 3′-ends of eukaryotic genes. This 3′-endsequence was homologous to other known omega-3 desaturases, sharing thehighest identity (41.5% in 130 amino acid overlap) with theSynechocystis delta-15 desaturase (Accession #D13780).

[0183] For the isolation of the 5′-end of the this gene, the followingoligonucleotides were designed and used in combinations with thepBluescript SK(+) vector oligonucleotide: RO 899: 5′-AGC GGA TAA CAA TTTCAC ACA GGA AAC AGC -3′. (SEQ. ID. NO: 17) RO 1185 (Reverse): 5′-GGT AAAAGA TCT CGT CCT TGT CGA TGT. (SEQ. ID. NO: 18) TGC-3′. RO 1186(Reverse): 5′-GTC AAA GTG GCT CAT CGT GC-3′. (SEQ.ID. NO: 19) RO 1187(Reverse): 5′-CGA GCG AGT ACG TGA GGT ACG CGT AC-3′. (SEQ. ID. NO: 20)

[0184] Amplifications were carried out using either the “Taq PCR MasterMix”-brand polymerase (Qiagen) or the “Advantage”-brand cDNA polymerase(Clonetech) or the “Platinum”-brand Taq DNA polymerase (LifeTechnologies), as described hereinabove for the 3′ end isolation.

[0185] PCR bands generated from primer combinations (RO 1185 or RO1186+RO 899) were between ˜580 to ˜440 bp and these were pooled andcloned into PCR-Blunt vector as described above. Clones thus generatedincluded “sdd17-14-1,” “sdd17-14-10,” “sdd17-18-2,” and “sdd17-18-8,”all of which showed homology with known omega-3 desaturases.

[0186] Additionally, bands generated from (RO 1187+RO 899) were ˜680 bp,and these were cloned separately to generate clones “sdd17-22-2” and“sdd17-22-5” which also showed homology with known omega-3 desaturases.All these clones overlapped with each other, as well as with theoriginal fragment of the unknown putative omega-3 desaturase. Thesesequences contained an ATG′ site followed by an open reading frame,indicating that it is the start site of this gene. These fragmentsshowed highest identity (39.7% in 146 amino acid overlap) with thedelta-15 desaturase from Calendula officinalis (Accession #AJ245938).

[0187] The full-length of this omega-3 desaturase was obtained by PCRamplification of the S. diclina cDNA library using the followingoligonucleotides:

[0188] RO 1212 (Forward): 5′-TCA ACA GAA TTC ATG ACC GAG GAT AAG ACG AAGGTC GAG TTC CCG-3′ (SEQ. ID. NO: 21). This primer contains the ‘ATG’start site (single underline) followed by the 5′ sequence of the omega-3desaturase. In addition, an EcoRI site (double underline) was introducedupstream of the start site to facilitate cloning into the yeastexpression vector pYX242.

[0189] RO 1213 (Reverse): 5′-AAA AGA AAG CTT CGC TTC CTA GTC TTA GTC CGACTT GGC CTT GGC-3′ (SEQ. ID. NO: 22). This primer contains the ‘TAA’stop codon (single underline) of the gene as well as sequence downstreamfrom the stop codon. This sequence was included because regions withinthe gene were very G+C rich, and thus could not be included in thedesign of oligonucleotides for PCR amplification. In addition, a HindIIIsite (double underline) was included for convenient cloning into a yeastexpression vector pYX242.

[0190] PCR amplification was carried out using the “Taq PCR MasterMix”-brand polymerase (Qiagen), 10 pmoles of each primer, and 1 μl ofthe cDNA library DNA from Example 1. Amplification was carried out asfollows: initial denaturation at 94° C. for 3 min, followed by 35 cyclesof the following: 94° C. for 1 min, 60° C. for 30 sec, 72° C. for 1 min.A final extension cycle of 72° C. for 7 min was carried out, followed bythe reaction termination at 4° C.

[0191] A PCR band of ˜1 kb was thus obtained and this band was isolated,purified, cloned into PCR-Blunt vector (Invitrogen), and transformedinto TOP10 cells. The inserts were sequenced to verify the genesequence. Clone “sdd17-27-2” was digested with EcoRI/HindIII to releasethe full-length insert, and this insert was cloned into yeast expressionvector pYX242, previously digested with EcoRI/HindIII. This constructcontained 1077 bp of sdd17 cloned into pYX242. This construct waslabeled pRSP19, which was transformed into yeast SC334 for expressionstudies.

[0192] In addition, the S. diclina omega-3 gene was cloned into anotheryeast expression vector, pYES2 (Invitrogen). For this, the omega-3desaturase gene was isolated from the cDNA library generated in Example1 by PCR amplification (as described above) using the followingoligonucleotides:

[0193] RO1221 (Forward) (SEQ. ID. NO: 23) 5′-TCA ACA AAG CTT ATG ACC GAGGAT AAG ACG AAG GTC GAG TTC CCG-3′. This primer contains the ‘ATG’ startsite (underlined) followed by the 5′ sequence of the omega-3 desaturase.In addition, a HindIII site (bold) was introduced upstream of the startsite to facilitate cloning into the pYES2 yeast expression vector.

[0194] RO1222 (Reverse) (SEQ. ID. NO: 24) 5′-AAA AGA GAA TTC CGC TTC CTAGTC TTA GTC CGA CTT GGC CTT GGC-3′ This primer contains the ‘TAA’ stopcodon (underlined) of the gene as well as sequence downstream from thestop codon. This sequence was included since regions within the genewere very G+C rich, and thus could not be included in the design ofoligonucleotides for PCR amplification. In addition, an EcoRI site(bold) was included for convenient cloning into the pYES2 yeastexpression vector.

[0195] The ˜1 kb PCR band thus generated was digested withHindIII/EcoRI, and cloned into pYES2 digested with the same restrictionenzymes. The resulting construct (sdd17+pYES2) was labeled pRSP20, andwas used in co-expression studies.

[0196] Attempts were also made to isolate the full-length sdd17 genefrom genomic DNA by PCR amplification. However, the PCR product obtainedwas larger than 1077 bp (˜1.15 kb), and sequencing of this productrevealed the presence of small introns in the genomic sequence.

[0197] The full-length gene of this putative omega-3 desaturase (labeledsdd17) was 1077 bp in length and is shown in FIG. 2 (SEQ ID NO: 25).

[0198] The gene of SEQ ID NO: 25 encoded a protein of 358 amino acidresidues (SEQ. ID. NO: 26) (FIG. 3). A search of the deduced proteinsequence of sdd17 (using the “tFastA” program) showed the protein tohave highest identity (41% in 269 amino acid overlap) with the delta-15desaturase from Synechocystis sp. (ATCC Accession No. 13780) (FIG. 4)and Synechocystis sp. PCC6803 (ATCC Accession No. D90913). This proteinshared sequence similarities with several other plant omega-3desaturases. Comparison of this predicted protein sequence with the FAT1enzyme from C. elegans (ATCC Accession L41807) revealed only a 31.6%identity in 310 amino acid overlap (FIG. 5).

[0199] Like all omega-3 desaturases, this enzyme does not contain acytochrome b5 domain within the 5′ end of its sequence. The cytochromeb5 domain is present in most “front-end” desaturating enzymes like delta5- and delta 6-desaturases. The omega-3 desaturase described in thisexample includes the three histidine-rich sequences that are present inall membrane-bound desaturases. These three domains are present atposition 89 to 93 (HDCGH), 125 to 129 (HRHHH), and 284 to 288 (HQVHH) ofSEQ. ID. NO: 26. These histidine-rich boxes are believed to co-ordinatethe diiron-oxo structure at the enzyme's active site, and are necessaryfor enzyme activity; see Stukey, J. E., McDonough, V. M. & Martn, C. E.(1990) J. Biol. Chem. 265, 20144-20149. These features are consistentwith the “SDD17” protein being a member of the membrane-bounddesaturase/hydroxylase family of the diiron-oxo proteins. The G+Ccontent of this gene is 61.8%.

EXAMPLE 5 Expression of the Omega-3 Desaturase Gene (“sdd17”) fromSaprolegnia diclina in Bakers' Yeast

[0200] To determine the substrate specificity and the class of reactioncatalyzed by the SDD17-protein, sdd17 was heterologously expressed in aSaccharomyces cerevisiae (SC334). Because S. cerevisiae cannotsynthesize fatty acids beyond OA (18:1n-9), it is an ideal system to useto determine enzyme activity on substrates longer than OA because nobackground enzyme activity will be detected. Suitable fatty acidsubstrates can be exogenously supplied to the host which are taken up bythe cell and acted on by the expressed protein of the transformed sdd17gene.

[0201] Clone pRSP19, which contained the full-length omega-3 desaturase(sdd17) from S. diclina cloned into pYX242, was transformed intoSaccharomyces cerevisiae (SC334) using the “Alkali-Cation YeastTransformation”-brand kit (BIO 101, Vista, Calif.). Transformants wereselected for leucine auxotrophy on media lacking leucine (DOB [-Leu]).To detect the specific desaturase activity of these clones,transformants were grown in the presence of 50 μM of each of LA(18:2n-6), GLA (18:3n-6), DGLA (20:3n-6), AA (20:4n-6), and adrenic acid(22:4n-6). Conversion of these exogenously supplied fatty acidsubstrates into a product having one additional unsaturation indicatesthe presence of a specific desaturase activity that is not found in thewild-type S. cerevisiae:

[0202] Conversion of LA (18:2n-6) to ALA (18:3n-3) indicates delta-15desaturase activity.

[0203] Conversion of GLA (18:3n-6) to STA(18:4n-3) indicates delta-15desaturase activity.

[0204] Conversion of DGLA (20:3n-6) to ETA (20:4n-3) indicates delta-17desaturase activity.

[0205] Conversion of AA (20:4n-6) to EPA (20:5n-3) indicates delta-17desaturase activity.

[0206] Conversion of adrenic acid (22:4n-6) to DPA (22:5n-3) indicatesdelta-19 desaturase activity.

[0207] The negative control strain was S. cerevisiae transformed withthe pYX242 vector. The experimental cultures and the control cultureswere grown simultaneously and analyzed.

[0208] The cultures were vigorously agitated (250 rpm) and grown for 48hours at 24° C. in the presence of 50 μM (final concentration) of thevarious substrates (see Table 3). The cells were spun down, washed oncein distilled water, and the pellets resuspended in methanol; chloroformwas added along with tritridecanoin (as an internal standard). Thesemixtures were incubated for at least an hour at room temperature, or at4° C. overnight. The chloroform layer was extracted and filtered througha Whatman filter with 1 gm anhydrous sodium sulfate to removeparticulate matter and residual water. The organic solvents wereevaporated at 40° C. under a stream of nitrogen. The extracted lipidswere then converted to fatty acid methyl esters (FAME) for gaschromatography analysis (GC) by adding 2 ml 0.5 N potassium hydroxide inmethanol to a closed tube. The samples were heated to 95° C.-10° C. for30 minutes and cooled to room temperature. Approximately 2 ml 14%borontrifluoride in methanol was added and the heating repeated. Afterthe extracted lipid mixture cooled, 2 ml of water and 1 ml of hexanewere added to extract the FAME for analysis by GC. The percentconversion was calculated using the formula: TABLE 3 Yeast Expression ofthe Omega- 3 Desaturase (SDD17) from Saprolegnia diclina at 24° C.Substrate* Product % Enzyme Clone Incorporated Produced conversionActivity PRSP19 18:2 n-6 18:3 n-3 0 Delta 15 (9.42%) (0%) 18:3 n-6 18:4n-3 0 Delta 15 (9.11%) (0%) 20:3 n-6 20:4 n-3 5% Delta 17 (21.36%)(1.18%) 20:4 n-6 20:5 n-3 13.8% Delta 17 (32.14%) (5.16%) 22:4 n-6 22:5n-3 4% Delta 19 (28.65%) (1.22%) Control 18:2 n-6 18:3 n-3 0 Delta 15(pYX242) (9.27%) (0%) 18:3 n-6 18:4 n-3 0 Delta 15 (9.18%) (0%) 20:3 n-620:4 n-3 0 Delta 17 (14.19%) (0%) 20:4 n-6 20:5 n-3 0 Delta 17 (26.56%)(0%) 22:4 n-6 22:5 n-3 0 Delta 19 (16.4%) (0%)${\% \quad {Conversion}} = {\frac{\left\lbrack {\% \quad {Product}} \right\rbrack}{\left\lbrack {{\% \quad {Product}} + {\% \quad {Substrate}}} \right\rbrack} \times 100}$

*50 μM substrate used Numbers in parenthesis represent fatty acid as apercentage of total lipids from yeast. 18:2n-6 = Linoleic acid 18:3n-6 =gamma-linolenic acid 18:3n-3 = alpha-linolenic acid 18:4n-3 =stearidonic acid 20:5n-3 = eicosapentaenoic acid 20:3n-6 =dihomo-gamma-linolenic acid 20:4n-6 = arachidonic acid 22:4n-6 = adrenicacid 20:4n-3 = eicosatetraenoic acid 22:5n-3 = omega-3-docosapentaenoicacid

[0209] Table 3 displays the enzyme activity of the sdd17-encoded proteinproduct from Saprolegnia diclina (ATCC 56851). This enzyme is an activeomega-3 desaturase capable of desaturating both C₂₀ and C₂₂ omega-6fatty acids substrates to yield the corresponding omega-3 fatty acidproducts. This enzyme converted 13.8% of the added AA substrate to thecorresponding EPA product, thus indicating delta-17 desaturase activity.In addition, this enzyme also acted on DGLA, converting it to ETA, aswould be expected for a delta-17 desaturase. In this Example, however,only 5% of the added DGLA was converted to ETA, indicating that underthe conditions used here, the enzyme has a substrate preference for AAas compared to DGLA.

[0210] The activity of this enzyme toward C₂₂ fatty acid substrates wasalso investigated because C₂₂ omega-3 fatty acids like DPA and DHA haveimportant dietary and pharmaceutical implications. From Table 3, it canbe seen that this enzyme was active on C₂₂ substrates such as adrenicacid, converting 4% of it to DPA. As can be seen from the controlcultures, there was no non-specific conversion of exogenously addedsubstrate to product in non-transformed S. cerevisiae.

[0211] Table 4 demonstrates that this omega-3 desaturase (SDD17) canalso function at a lower temperature. (i.e. 15° C.). Here, 50 μM ofexogenous substrate was added to the transformants and the cultures weregrown for 48 hours at 15° C. Fatty acid analysis was carried out asdescribed above. The overall uptake of substrate by S. cerevisiae at 15°C. was lower than that seen at 24° C. (compare Table 3 & Table 4).However the percent conversion of substrate to product by the enzymeincreased at 15° C. Since the presence of lower concentration ofexogenous fatty acid substrate seemed to improve the activity of theenzyme, it is possible that fatty acid substrates at high concentrationsmay exert a feed-back inhibition on this enzyme. Further studies may becarried out to determine the effect of different substrate concentrationand different temperatures on expression of this omega-3 desaturase inS. cerevisiae. TABLE 4 Yeast Expression of the Omega-3 Desaturase(SDD17) from Saprolegnia diclina at 15° C. Substrate* Product % EnzymeClone Incorporated Produced Conversion Activity PRSP19 18:2 n-6 18:3 n-30 Delta 15 (8.79%) (0%) 18:3 n-6 18:4 n-3 0 Delta 15 (12.69%) (0%) 20:3n-6 20:4 n-3  13% Delta 17 (9.52%) (1.46%) 20:4 n-6 20:5 n-3  19% Delta17 (8.69%) (2.05%) 22:4 n-6 22:5 n-3 8.4% Delta 19 (6.68%) (0.62%)Control 18:2 n-6 18:3 n-3 0 Delta 15 (pYX242) (9.65%) (0%) 18:3 n-6 18:4n-3 0 Delta 15 (13.55%) (0%) 20:3 n-6 20:4 n-3 0 Delta 17 (10.17%) (0%)20:4 n-6 20:5 n-3 0 Delta 17 (16.58%) (0%) 22:4 n-6 22:5 n-3 0 Delta 19(11.05%) (0%)

[0212] Unlike all known omega-3 desaturases, the sdd17-encoding enzymedid not desaturate any C₁₈ omega-6 fatty acyl substrates to theircorresponding omega-3 fatty acids (under the conditions tested). It ispossible that in vivo, this enzyme functions exclusively on AA,converting it to EPA. This would be consistent with the fatty acidprofile of S. diclina displaying high amounts of AA and EPA, but littleor none of the other omega-3 intermediates (Table 5). TABLE 5 Fatty AcidProfile of Saprolegnia diclina ATCC 56851 Fatty Acid % Total Lipid C10:00.22 C12:0 0.1 C13:0 3.98 C14:0 6.0 C14:1 n-5 0.29 C15:0 1.06 C16:019.75 C16:1 n-7 1.99 C18:0 3.99 C18: n-9 15.39 C18:1 n-7 7.39 C18:1 n-50.43 C18:2 n-6 7.07 C18:3 n-6 2.13 C18:3 n-3 0.08 C20:0 0.76 C20:1 n-90.15 C20:1 n-7 0.08 C20:2 n-6 0.22 C20:3 n-6 1.31 C20:4 n-6 15.42 C20:5n-3 12.2

[0213] Thus, sdd17 encodes a novel omega-3 desaturase, capable ofdesaturating C₂₀ and C₂₂ fatty acid substrates. The SDD17 protein caneasily be expressed in a heterologous system and thus has potential foruse in other heterologous systems like plants. This enzyme is verydifferent from other known omega-3 desaturases, showing activity on bothC₂₀ and C₂₂ fatty acid substrates, but not C₁₈ substrates. It sharesonly 31.6% identity with FAT-1, the only other known desaturase capableof acting on C₂₀ and C₂₂ omega-6 fatty acid substrates. Thus the enzymeencoded by sdd17 is a novel omega-3 desaturase.

EXAMPLE 6 Co-Expression of S. diclina Omega-3 Desaturase with OtherEnzymes

[0214] The pRSP20 construct consisting of sdd17 cloned into pYES2 yeastexpression vector, as described in Example 3, was used in co-expressionstudies with other desaturases and elongases. pRSP3, a construct thatcontained the delta 5-desaturase gene (SEQ ID NO: 27) from S. diclinacloned into the pYX242 yeast expression vector, was co-transformed withpRSP20 into yeast. Transformation protocol was as described in Example4. This delta 5-desaturase catalyzes the conversion of DGLA to AA andETA to EPA. Co-transformants were selected on minimal media lackingleucine and Uracil (DOB [-Leu-Ura]).

[0215] Table 6 shows that when 50 μM of the substrate DGLA (20:3 n-6)was added, the delta 5-desaturase converted it to AA (20:4, n-6), andthe omega-3 desaturase was able to further desaturate AA to EPA (24:5,n-3). The percent conversion of the substrate to final product was 5%,with no background observed in the negative control. TABLE 6Co-expression Studies with the Omega -3 Desaturase (SDD17) from S.diclina 20:3 n-6 20:4 n-6 20:5 n-3 Plasmid in (DGLA) (AA) (EPA) % Cloneyeast Incorporated Produced Produced Conversion Cntrl pYX242 + 19.33 0 00 pYES2 pRSP22 pRSP3 (Delta 20.56 2.64 0.14 5% 5) + pRSP20 (omega -3desaturase) 18:3 n-6 20:3 n-6 20:4 n-3 Plasmid in (GLA) (DGLA) (ETA) %Clone yeast Incorporated Produced Produced Conversion Cntrl pYX242 +4.83 0 0 0 pYES2 pRSP23 pRAT-4-A7 4.56 9.30 0.14 1.4% (elongase) +pRSP20 (omega-3 desaturase) * 50 μM substrate used Numbers representfatty acid as a percentage of total lipids from yeast 18:3n-6 =gamma-linolenic acid 20:3n-6 = dihomo-gamma-linolenic acid 20:4n-6 =arachidonic acid 20:4n-3 = eicosatetraenoic acid 20:5n-3 =eicosapentaenoic acid${\% \quad {Conversion}} = \frac{\left\lbrack {{\% \quad {Product}\quad 1} + {\% \quad {Product}\quad 2}} \right\rbrack}{\left\lbrack {{\% \quad {substrate}} + {\% \quad {Product}\quad 1} + {\% \quad {Product}\quad 2}} \right\rbrack}$

[0216] Table 6 also shows the results of a co-transformation experimentinvolving pRSP20 and pRAT-4-A7, an elongase obtained fromThraustochytrid sp. 7091 (SEQ. ID. NO: 28) cloned into pYX242. Thiselongase gene catalyzes the addition of two more carbons to thepre-existing PUFA. When 50 μM of the substrate GLA (18:3 n-6) was addedto the co-transformants, the elongase elongated GLA to DGLA, and theDGLA was further desaturated by the omega-3 desaturase to ETA (20:4n-3). The percent conversion of substrate to final product was 1.4%,with no background observed in the negative control.

[0217] Thus the S. diclina omega-3 desaturase was able to utilize aproduct produced, in a heterologous expression system, by anotherheterologous enzyme from the PUFA biosynthetic pathway, and convert thatproduct to the expected PUFA.

[0218] It should be noted that the expression (and hence the activity)of sdd17 when cloned in the pYES2 vector (pRSP20) was much lower thanwhen cloned into the pYX242 vector (pRSP19). This could be explained bythe difference in the expression promoters present in each vector. ThepYX242 promoter is a constitutive promoter and is much stronger than thegalactose-inducible promoter in pYES2. Similar observations have beenmade during expression studies with other desaturases when cloned intothese two expression vectors.

[0219] Further co-expression studies may be carried out using pRSP19instead of pRSP20 along with other desaturases and elongases. Also theS. diclina omega-3 desaturase may also be co-expressed with otherenzymes like the delta 4-desaturase pRTA7 (SEQ. ID. NO: 29), where inadrenic acid (22:4 n-G) may be added as a substrate and the final endproduct DHA (22:6 n-3) may be produced due to the concerted action ofthe omega-3 desaturase and the delta 4-desaturase.

EXAMPLE 7 Design of Degenerate Primers for the Isolation of the Delta12-Desaturase Gene from Saprolegnia diclina ATCC 56851

[0220] Analysis of the fatty acid composition of Saprolegnia diclina(ATCC 56851) revealed the presence of a considerable amount of LA, whichsuggested the presence of a very active delta 12-desaturase (Table 5).Delta 12-desaturases use OA as a substrate, thus catalyzing theconversion of OA to LA (see FIG. 1). Delta 12-desaturases are presentonly in plants, fungi, and insects, but not in mammals, includinghumans. Thus LA is an “essential” fatty acid in humans because it cannotbe synthesized in vivo. LA is further desaturated and elongated toproduce important intracellular compounds like GLA, AA, and EPA.

[0221] The goal of this experiment was to isolate the delta12-desaturase gene from S. diclina and verify its functionality byexpressing the enzyme in a heterologous host system such as yeast. Theapproach taken was to design degenerate primers (oligonucleotides) thatrepresent conserved amino acid motifs from known delta 12-desaturases.In designing these primers, known delta-12 desaturase sequenceinformation from both fungi and plant sources was used, includingsequence information from: Mortierella alpina (Accession #AF110509),Mucor rouxii (Accession #AF161219), Brassica juncea (Accession #X91139),Arabidopsis thaliana (Accession #L26296), and Borago officinalis(Accession #AF0744324). The sequence information was analyzed using theCODEHOP Blockmaker program (http://blocks.fhcrc.org/codehop.html).

[0222] The degenerate primers used in this Example were as follows:Protein Motif 1: NH₃— P N/E FTIKEIR D/E A/C IPAHCF —COOH Primer RO 967(Forward): 5′-CCG SAG TTC ACS ATC AAG GAG ATC (SEQ. ID. NO: 30) CGC GASKSC ATC CCG GCC CAC TGC TTC-3′. Protein Motif 2: NH₃— MP H/F YHAEEAT V/YH I/L KK A/L —COOH Primer RO 968 (Reverse): 5′-GRS CTT CTT GAK GTG GWMSGT GGC (SEQ. ID. NO: 31) CTC CTC GGC GTG GTA GWR CGG CAT-3′. ProteinMotif 3: NH₃— P L/V YW A/I C/M/A QG V/I V L/G/C TGVW —COOH Primer RO 964(Forward): 5′-CCS STC TAC TGG GCC TGC CAG GGT (SEQ. ID. NO: 32) RTC GTCCTC ACS GGT GTC TGG-3′.

[0223] This sequence is more similar to the known plant Delta12-desaturases. Primer RO 965 (Forward): 5′-CCS STC TAC TGG ATC RYS CAGGGT (SEQ. ID. NO: 33) RTC GTC KGY ACS GGT GTC TGG-3′.

[0224] This sequence is more similar to the known fungal Delta12-desaturases. Protein Motif 4: NH₃— HVAHH L/F FS T/Q MPHYHA —COOHPrimer RO 966 (Reverse): 5′-GGC GTG GTA GTG CGG CAT SMM CGA (SEQ. ID.NO: 34) GAA GAR GTG GTG GGC GAC GTG-3′.

[0225] The degeneracy code used for the oligonucleotides was as follows:R=A/G; Y=C/T; M=A/C; K=G/T; W=A/T; S=C/G; B=C/G/T; D=A/G/T; H=A/C/T;V=A/C/G; N=A/C/G/T.

EXAMPLE 8 Identification and Isolation of the Delta 12-Desaturase Genefrom Saprolegnia diclina (ATCC 56851)

[0226] To isolate a fragment of the delta 12-desaturase gene from S.diclina, PCR was carried out using the S. diclina cDNA library fromExample 1 as a template. Primers were used in the followingcombinations: (RO 964+RO 966), (RO 965+RO 966), and (RO 967+RO 968). PCRwas carried out in 100 μl volumes using the “Taq PCR Master Mix”-brandpolymerase (Qiagen). 10 pmoles of each primer were used along with 1 μlof the cDNA library DNA. Amplification was carried out as follows:initial denaturation at 94° C. for 4 min, followed by 25 cycles of thefollowing: 94° C. for 1 min, 47° C. for 1 min, 72° C. for 2 min. A finalextension cycle of 72° C. for 5 min was carried out, followed byreaction termination at 4 C.

[0227] Amplification with (RO 964+RO 966) or (RO 965+RO 966) resulted indistinct bands of ˜688 bp in length. Amplification with (RO 967+RO 968)resulted in one distinct band of ˜660 bp. These bands were resolved on a“SeaKem Gold”-brand agarose gel (FMC BioProducts), and gel-purifiedusing the Qiagen Gel Extraction Kit. The staggered ends on the fragmentwere “filled-in” using T4 DNA polymerase (Life Technologies), followingthe manufacturer's specifications. The DNA fragments were then clonedinto the PCR-Blunt vector (Invitrogen). The recombinant plasmids weretransformed into TOP10 supercompetent cells (Invitrogen), clones weresequenced, and a database search (Gen-EMBL) was carried out.

[0228] Clones “sdd12-1-8,” “sdd12-2-8,” and “sdd12-5-1” were all foundto overlap with each other, and these overlapping fragments were alignedusing the “ASSEMBLE”-brand program (GCG) to create a single fusionfragment of ˜900 bp. A “tFastA” search with the deduced amino acids ofthis fusion sequence showed highest identity to the following proteins:

[0229] 49% identity in 298 amino acid overlap with Borago officinalisDelta 12-desaturase (Accession #AF074324) and 46.7% identity in 332amino acid overlap with Sesamum indicum Delta 12-desaturase (Accession#AF192486).

[0230] Based on the high identity to known delta 12-desaturases, thefragment was considered to be a region of the S. diclina delta12-desaturase gene. This fragment was used to design primers to pull upthe 5′- and 3′-ends of the gene.

[0231] To isolate the 3′ end of the gene, the following oligonucleotideswere designed: RO 975 (Forward): 5′-CAC GTA CCT CCA GCA CAC GGA CAC CTACG-3′. (SEQ. ID. NO: 35) RO 976 (Forward): 5′-GAT CGA CAG CGC GAT CCACCA CAT TGC-3′. (SEQ. ID. NO: 36)

[0232] These were used in combinations with the pBluescript SK(+) vectoroligonucleotide RO 898: 5′-CCC AGT CAC GAC GTT GTA AAA CGA CGG CCA G-3′(SEQ. ID. NO: 16).

[0233] PCR amplifications were carried out using “Taq PCR MasterMix”-brand polymerase (Qiagen) as follows: 10 pmoles of each primer wereused along with 1 μl of the cDNA library DNA from Example 1 as template.Amplification conditions were as follows: initial denaturation at 94° C.for 3 min, followed by 35 cycles of the following: 94° C. for 1 min, 60°C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for 7min was carried out, followed by reaction termination at 4° C.

[0234] Primer combination (RO 898+RO 975) generated a PCR band of 390 bpand primer combination (RO 898+RO 976) generated a band of length ˜300bp. These bands were purified and cloned into PCR-Blunt vector asdescribed earlier. Several clones, including clones “sdd12-8-12” and“sdd12-9-4” were found to contain the 3′ end of the delta 12-desaturasegene. These sequences overlapped the initial delta-12 desaturasefragment and included a TAA stop codon and a poly-A tail. Sequenceanalysis with the “tFastA” program revealed that clone “sdd12-9-4”shared 54.5% identity in a 73 amino acid overlap with the delta12-desaturase from Mortierella alpina (Accession #AB020033), and 56.9%identity in a 72 amino acid overlap with the delta 12-desaturase fromMucor rouxii (Accession #AF161219).

[0235] To isolate the 5′ end of the this gene, the followingoligonucleotides were designed and used in combinations with thepBluescript SK(+) vector oligonucleotide RO 899 (SEQ. ID. NO: 17). RO977 (Reverse): 5′-CAA ATG GTA AAA GCT AGT GGC AGC GCT GC-3′. (SEQ ID NO:37) RO 978 (Reverse): 5′-AGT ACG TGC CCT GGA CGA ACC AGT AGA TG-3′. (SEQID NO: 38)

[0236] PCR amplifications were carried out using either “Taq PCR MasterMix”-brand polymerase (Qiagen) or the “Advantage-GC”-brand cDNA PCR kit(Clonetech). The Clonetech product was used to circumvent potential PCRamplification problems that may occur with GC-rich regions generallypresent at the 5′-end of desaturases from this organism. PCRamplifications using the “Taq PCR Master Mix”-brand polymerase wascarried out as described for the isolation of 3′-end of this gene.

[0237] When using the “Advantage-GC cDNA PCR”-brand kit, thermocyclingconditions were as follows: the template was initially denatured at 94°C. for 1 min, followed by 30 cycles of 94° C. for 30 seconds, 68° C. for3 minutes, and finally an extension cycle at 68° C. for 5 min. Eachreaction included 1 μl of cDNA library template (from Example 1), 10pmole of each primer, 0.2 mM each dNTP, 1M GC Melt, 40 mM Tricine-KOH,15 mM KOAc, 3.5 mM MG(OAc)₂, 5% DMSO, and 375 μg/ml BSA in a totalvolume of 50 μl.

[0238] A PCR product of ˜371 bp was obtained using the primercombination (RO 899+RO 978). This band was cloned into the PCR-Bluntvector (Invitrogen) as described earlier. Only one clone, “sdd12-10-8,”thus obtained contained the putative ATG start site of the gene. Otherclones had the ATG replaced by other codons. “tFastA” analysis of thededuced amino acid sequence of “sdd12-10-8” showed 47.2% identity in a72 amino acid overlap of the delta-12 desaturase from ˜Impatiensbalsamina (Accession #AF182520) and 42.7% identity in a 75 amino acidoverlap with the delta-12 desaturase from Calendula officinalis(Accession #AJ245938).

[0239] The full length of this delta 12-desaturase was obtained by PCRamplification of the S. diclina cDNA library of Example 1 using thefollowing oligonucleotides:

[0240] RO1051 (Forward): 5′-TCA ACA GAA TTC ATG TGC AAA GGT CAA GCT CCTTCC AAG GCC GAC GTG-3′ (SEQ. ID. 39). This primer contains the ‘ATG’start site (underlined) followed by the 5′ sequence of the Delta12-desaturase. In addition, an EcoRI site (double underline) wasintroduced upstream of the start site to facilitate cloning into thepYX242 yeast expression vector.

[0241] RO1057 (Reverse): 5′-AAA AGA AAG CTT TTA CTT TTC CTC GAG CTT GCGCTT GTA AAA CAC AAC-3′ (SEQ. ID. NO: 40). This primer contains the TAAstop codon (underlined) of the gene as well as a HindIII site (doubleunderline), which was included for convenient cloning into the pYX242yeast expression vector.

[0242] PCR amplifications were carried out using both “Taq PCR MasterMix”-brand polymerase (Qiagen) and the “Advantage-GC cDNA PCR”-brand kit(Clonetech), as described earlier. In this case, however, S. diclinagenomic DNA was used as the template for amplification. A PCR band of˜1.1 kb was thus obtained and this band was isolated, purified, clonedinto PCR-Blunt vector (Invitrogen), and transformed into TOP10 cells.The inserts were sequenced to verify the gene sequence. Clone“sdd12-gg-b1” was digested with EcoRI/HindIII to release the full-lengthinsert, and this insert was cloned into the yeast expression vectorpYX242, previously digested with EcoRI/HindIII. This construct included1182 bp of the delta-12 desaturase gene and pYX242. The construct waslabeled pRSP11. The pRSP11 construct was then transformed into S.cerevisiae (SC334) for expression studies.

[0243] The full-length gene of this putative delta-12 desaturase(labeled sdd12) was 1182 bp in length (SEQ ID NO: 41) (FIG. 6). The geneencodes a protein of 393 amino acid residues (SEQ ID NO: 42) (FIG. 7). A“tFastA” search of the deduced protein sequence of sdd12 showed theprotein to have highest identity (45.6% in a 379 amino acid overlap)with the delta-12 desaturase from Gossypium hirsutum (Accession #X97016)(FIG. 8) and 49.6% identity in a 353 amino acid overlap with thedelta-12 desaturase from Sesamum indicum (FAD2) (Accession #AF192486).

[0244] Like other delta-12 desaturases, this enzyme also does notcontain a cytochrome b5 domain within the 5‘end of its’ sequence. Thisenzyme does contain the three histidine-rich sequences that are presentin all membrane-bound desaturases. The position and length of thesehistidine-boxes are similar to those seen in other desaturases. Theseare present at amino acid positions 108 to 112 (HECGH), 144 to 148(HRRHH), and 326 to 330 (HVTHH) of SEQ. ID. NO: 42. As noted earlier,these histidine-rich boxes are believed to co-ordinate the diiron-oxostructure at the enzyme's active site and are necessary for enzymeactivity.

EXAMPLE 9 Expression of the Delta 12-Desaturase Gene (sdd12) in Bakers'Yeast

[0245] To determine the substrate specificity and the class of reactioncatalyzed by this delta 12-desaturase (SDD12), sdd12 was heterologouslyexpressed in a Saccharomyces cerevisiae (SC334). As noted earlier,because S. cerevisiae cannot synthesize fatty acids beyond OA, it is anideal system to determine enzyme activity on substrates longer than OAbecause no background enzyme activity will be detected. Suitable fattyacid substrates are exogenously supplied to the host; these substratesare taken up by the cell and acted on by the expressed delta-12desaturase of the transformed sdd12 gene.

[0246] Clone pRSP11, which contained the full-length delta-12 desaturase(sdd12) from S. diclina, cloned into pYX242, was transformed intoSaccharomyces cerevisiae (SC334) using the “Alkali-Cation YeastTransformation”-brand kit (BIO 101), following the manufacturer'sinstructions. Transformants were selected for leucine auxotrophy onmedia lacking leucine (DOB-Leu). To detect the specific desaturaseactivity of these clones, transformants were grown in the presence of 50μM each of OA, LA, GLA, and DGLA.

[0247] Conversion of OA to LA (18:2n-6) indicates delta-12 desaturaseactivity.

[0248] Conversion of LA to ALA (18:3n-3) indicates delta-15 desaturaseactivity.

[0249] Conversion of LA to GLA (18:3n-6) indicates delta-6 desaturaseactivity.

[0250] Conversion of GLA to stearidonic (18:4n-3) acid indicatesdelta-15 desaturase activity.

[0251] Conversion of DGLA to ETA (20:4n-3) indicates delta-17 desaturaseactivity.

[0252] Conversion of DGLA to AA (20:4n-6) indicates delta-5 desaturaseactivity.

[0253] The negative control strain was S. cerevisiae transformed withthe pYX242 vector. The experimental and control cultures were grownsimultaneously and analyzed.

[0254] The cultures were vigorously agitated (250 rpm) and grown for 48hours at 24° C. in the presence of 50 μM (final concentration) of thevarious substrates (see Table 7). The cells were spun down, washed oncein distilled water, and the pellets resuspended in methanol; chloroformwas added along with tritridecanoin (as an internal standard). Thesemixtures were incubated for at least an hour at room temperature, or at4° C. overnight. The chloroform layer was extracted and filtered througha Whatman filter with 1 gm anhydrous sodium sulfate to removeparticulate matter and residual water. The organic solvents wereevaporated at 40° C. under a stream of nitrogen. The extracted lipidswere then converted to fatty acid methyl esters (FAME) for gaschromatography analysis (GC) by adding 2 ml 0.5 N potassium hydroxide inmethanol to a closed tube. The samples were heated to 95° C.-100° C. for30 minutes and cooled to room temperature. Approximately 2 ml 14%borontrifluoride in methanol was added and the heating repeated. Afterthe extracted lipid mixture cooled, 2 ml of water and 1 ml of hexanewere added to extract the FAME for analysis by GC. The percentconversion was calculated using the formula:${\% \quad {Conversion}} = {\frac{\left\lbrack {\% \quad {Product}} \right\rbrack}{\left\lbrack {{\% \quad {Product}} + {\% \quad {Substrate}}} \right\rbrack} \times 100}$

[0255] Table 7 shows the enzyme activity of the delta-12 desaturase whenexpressed in yeast. Here, the pRSP11 clone when expressed was capable ofconverting 35.8% of OA substrate to LA, indicating delta-12 desaturaseactivity.

[0256] In Table 7, the fatty acids of interest are represented as apercentage of the total lipids extracted from yeast. GC/MS was employedto identify the products. Under these conditions, the clones did notexhibit other desaturase activities. This confirmed that the geneisolated is a delta-12 desaturase gene. No background substrateconversion was detected using hosts transformed with just the vectoralone. This data indicates that this delta-12 desaturase can beexpressed in a heterologous system and is thus useful in the productionof transgenic polyunsaturated fatty acids like GLA, AA, EPA and DHA.TABLE 7 Saprolegnia diclina Delta 12-Desaturase Expression in Baker'sYeast at 24° C. % Desatur. Substrate* Product Conversion Clone ActivityIncorporated Produced of Substrate pRSP11 Delta OA (17.09%) LA (9.59%)35.8% (pYX242 + 12 Delta 12- Delta LA (18.14%) ALA (0.06%) 0 Desaturase15 (S. Delta LA (18.14%) GLA (0%) 0 diclina )) 6 Delta 5 DGLA (25.38%)AA (0.17%) 0 Delta DGLA (25.38%) ETA (0.07%) 0 17 Control Delta OA(18.99%) LA (0.09%) 0 (pYX242) 12 Delta LA (8.63%) ALA (0%) 0 15 DeltaLA (8.63%) GLA (0%) 0 6 Delta DGLA (13.74%) AA (0%) 0 5 Delta DGLA(13.74%) ETA (0%) 0 17

[0257] Nutritional Compositions

[0258] The PUFAs described in the Detailed Description may be utilizedin various nutritional supplements, infant formulations, nutritionalsubstitutes and other nutritional solutions.

[0259] I. Infant Formulations

[0260] A. Isomil® Soy Formula with Iron:

[0261] Usage: As a beverage for infants, children and adults with anallergy or sensitivity to cows milk. A feeding for patients withdisorders for which lactose should be avoided: lactase deficiency,lactose intolerance and galactosemia.

[0262] Features:

[0263] Soy protein isolate to avoid symptoms of cow's-milk-proteinallergy or sensitivity.

[0264] Lactose-free formulation to avoid lactose-associated diarrhea.

[0265] Low osmolality (240 mOs/kg water) to reduce risk of osmoticdiarrhea.

[0266] Dual carbohydrates (corn syrup and sucrose) designed to enhancecarbohydrate absorption and reduce the risk of exceeding the absorptivecapacity of the damaged gut.

[0267] 1.8 mg of Iron (as ferrous sulfate) per 100 Calories to helpprevent iron deficiency.

[0268] Recommended levels of vitamins and minerals.

[0269] Vegetable oils to provide recommended levels of essential fattyacids.

[0270] Milk-white color, milk-like consistency and pleasant aroma.

[0271] Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6% sugar(sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4% coconut oil,0.15% calcium citrate, 0.11% calcium phosphate tribasic, potassiumcitrate, potassium phosphate monobasic, potassium chloride, mono- anddisglycerides, soy lecithin, carrageenan, ascorbic acid, L-methionine,magnesium chloride, potassium phosphate dibasic, sodium chloride,choline chloride, taurine, ferrous sulfate, m-inositol, alpha-tocopherylacetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate,cupric sulfate, vitamin A palmitate, thiamine chloride hydrochloride,riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate,potassium iodide, phylloquinone, biotin, sodium selenite, vitamin D3 andcyanocobalamin.

[0272] B. Isomil® DF Soy Formula For Diarrhea:

[0273] Usage: As a short-term feeding for the dietary management ofdiarrhea in infants and toddlers.

[0274] Features:

[0275] First infant formula to contain added dietary fiber from soyfiber specifically for diarrhea management.

[0276] Clinically shown to reduce the duration of loose, watery stoolsduring mild to severe diarrhea in infants.

[0277] Nutritionally complete to meet the nutritional needs of theinfant.

[0278] Soy protein isolate with added L-methionine meets or exceeds aninfant's requirement for all essential amino acids.

[0279] Lactose-free formulation to avoid lactose-associated diarrhea.

[0280] Low osmolality (240 mOsm/kg water) to reduce the risk of osmoticdiarrhea.

[0281] Dual carbohydrates (corn syrup and sucrose) designed to enhancecarbohydrate absorption and reduce the risk of exceeding the absorptivecapacity of the damaged gut.

[0282] Meets or exceeds the vitamin and mineral levels recommended bythe Committee on Nutrition of the American Academy of Pediatrics andrequired by the Infant Formula Act.

[0283] 1.8 mg of iron (as ferrous sulfate) per 100 Calories to helpprevent iron deficiency.

[0284] Vegetable oils to provide recommended levels of essential fattyacids.

[0285] Ingredients: (Pareve) 86% water, 4.8% corn syrup, 2.5% sugar(sucrose), 2.1% soy oil, 2.0% soy protein isolate, 1.4% coconut oil,0.77% soy fiber, 0.12% calcium citrate, 0.11% calcium phosphatetribasic, 0.10% potassium citrate, potassium chloride, potassiumphosphate monobasic, mono and diglycerides, soy lecithin, carrageenan,magnesium chloride, ascorbic acid, L-methionine, potassium phosphatedibasic, sodium chloride, choline chloride, taurine, ferrous sulfate,m-inositol, alpha-tocopheryl acetate, zinc sulfate, L-carnitine,niacinamide, calcium pantothenate, cupric sulfate, vitamin A palmitate,thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride,folic acid, manganese sulfate, potassium iodide, phylloquinone, biotin,sodium selenite, vitamin D3 and cyanocobalamin.

[0286] C. Isomil® SF Sucrose-Free Soy Formula with Iron:

[0287] Usage: As a beverage for infants, children and adults with anallergy or sensitivity to cow's-milk protein or an intolerance tosucrose. A feeding for patients with disorders for which lactose andsucrose should be avoided.

[0288] Features:

[0289] Soy protein isolate to avoid symptoms of cow's-milk-proteinallergy or sensitivity.

[0290] Lactose-free formulation to avoid lactose-associated diarrhea(carbohydrate source is Polycose® Glucose Polymers).

[0291] Sucrose free for the patient who cannot tolerate sucrose.

[0292] Low osmolality (180 mOsm/kg water) to reduce risk of osmoticdiarrhea.

[0293] 1.8 mg of iron (as ferrous sulfate) per 100 Calories to helpprevent iron deficiency.

[0294] Recommended levels of vitamins and minerals.

[0295] Vegetable oils to provide recommended levels of essential fattyacids.

[0296] Milk-white color, milk-like consistency and pleasant aroma.

[0297] Ingredients: (Pareve) 75% water, 11.8% hydrolized cornstarch,4.1% soy oil, 4.1% soy protein isolate, 2.8% coconut oil, 1.0% modifiedcornstarch, 0.38% calcium phosphate tribasic, 0.17% potassium citrate,0.13% potassium chloride, mono- and diglycerides, soy lecithin,magnesium chloride, abscorbic acid, L-methionine, calcium carbonate,sodium chloride, choline chloride, carrageenan, taurine, ferroussulfate, m-inositol, alpha-tocopheryl acetate, zinc sulfate,L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitaminA palmitate, thiamine chloride hydrochloride, riboflavin, pyridoxinehydrochloride, folic acid, manganese sulfate, potassium iodide,phylloquinone, biotin, sodium selenite, vitamin D3 and cyanocobalamin.

[0298] D. Isomil® 20 Soy Formula With Iron Ready To Feed, 20 Cal/fl oz.:

[0299] Usage: When a soy feeding is desired.

[0300] Ingredients: (Pareve) 85% water, 4.9% corn syrup, 2.6%sugar(sucrose), 2.1% soy oil, 1.9% soy protein isolate, 1.4% coconutoil, 0.15% calcium citrate, 0.11% calcium phosphate tribasic, potassiumcitrate, potassium phosphate monobasic, potassium chloride, mono- anddiglycerides, soy lecithin, carrageenan, abscorbic acid, L-methionine,magnesium chloride, potassium phosphate dibasic, sodium chloride,choline chloride, taurine, ferrous sulfate, m-inositol, alpha-tocopherylacetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate,cupric sulfate, vitamin A palmitate, thiamine chloride hydrochloride,riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate,potassium iodide, phylloquinone, biotin, sodium selenite, vitamin D3 andcyanocobalamin.

[0301] E. Similac® Infant Formula:

[0302] Usage: When an infant formula is needed: if the decision is madeto discontinue breastfeeding before age 1 year, if a supplement tobreastfeeding is needed or as a routine feeding if breastfeeding is notadopted.

[0303] Features:

[0304] Protein of appropriate quality and quantity for good growth;heat-denatured, which reduces the risk of milk-associated enteric bloodloss.

[0305] Fat from a blend of vegetable oils (doubly homogenized),providing essential linoleic acid that is easily absorbed.

[0306] Carbohydrate as lactose in proportion similar to that of humanmilk.

[0307] Low renal solute load to minimize stress on developing organs.

[0308] Powder, Concentrated Liquid and Ready To Feed forms.

[0309] Ingredients: (-D) Water, nonfat milk, lactose, soy oil, coconutoil, mono- and diglycerides, soy lecithin, abscorbic acid, carrageenan,choline chloride, taurine, m-inositol, alpha-tocopheryl acetate, zincsulfate, niacinamide, ferrous sulfate, calcium pantothenate, cupricsulfate, vitamin A palmitate, thiamine chloride hydrochloride,riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate,phylloquinone, biotin, sodium selenite, vitamin D3 and cyanocobalamin.

[0310] F. Similac® NeoCare Premature Infant Formula With Iron:

[0311] Usage: For premature infants' special nutritional needs afterhospital discharge. Similac NeoCare is a nutritionally complete formuladeveloped to provide premature infants with extra calories, protein,vitamins and minerals needed to promote catch-up growth and supportdevelopment.

[0312] Features:

[0313] Reduces the need for caloric and vitamin supplementation. Morecalories (22 Cal/fl oz) than standard term formulas (20 Cal/fl oz).

[0314] Highly absorbed fat blend, with medium-chain triglycerides(MCToil) to help meet the special digestive needs of premature infants.

[0315] Higher levels of protein, vitamins and minerals per 100 caloriesto extend the nutritional support initiated in-hospital.

[0316] More calcium and phosphorus for improved bone mineralization.

[0317] Ingredients: -D Corn syrup solids, nonfat milk, lactose, wheyprotein concentrate, soy oil, high-oleic safflower oil, fractionatedcoconut oil (medium chain triglycerides), coconut oil, potassiumcitrate, calcium phosphate tribasic, calcium carbonate, ascorbic acid,magnesium chloride, potassium chloride, sodium chloride, taurine,ferrous sulfate, m-inositol, choline chloride, ascorbyl palmitate,L-carnitine, alpha-tocopheryl acetate, zinc sulfate, niacinamide, mixedtocopherols, sodium citrate, calcium pantothenate, cupric sulfate,thiamine chloride hydrochloride, vitamin A palmitate, beta carotene,riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate,phylloquinone, biotin, sodium selenite, vitamin D3 and cyanocobalamin.

[0318] G. Similac Natural Care Low-Iron Human Milk Fortifier Ready ToUse, 24 Cal/fl oz.:

[0319] Usage: Designed to be mixed with human milk or to be fedalternatively with human milk to low-birth-weight infants.

[0320] Ingredients: -D Water, nonfat milk, hydrolyzed cornstarch,lactose, fractionated coconut oil (medium-chain triglycerides), wheyprotein concentrate, soy oil, coconut oil, calcium phosphate tribasic,potassium citrate, magnesium chloride, sodium citrate, ascorbic acid,calcium carbonate, mono and diglycerides, soy lecithin, carrageenan,choline chloride, m-inositol, taurine, niacinamide, L-carnitine, alphatocopheryl acetate, zinc sulfate, potassium chloride, calciumpantothenate, ferrous sulfate, cupric sulfate, riboflavin, vitamin Apalmitate, thiamine chloride hydrochloride, pyridoxine hydrochloride,biotin, folic acid, manganese sulfate, phylloquinone, vitamin D3, sodiumselenite and cyanocobalamin.

[0321] Various PUFAs of this invention can be substituted and/or addedto the infant formulae described above and to other infant formulaeknown to those in the art.

[0322] II. Nutritional Formulations

[0323] A. ENSURE®

[0324] Usage: ENSURE is a low-residue liquid food designed primarily asan oral nutritional supplement to be used with or between meals or, inappropriate amounts, as a meal replacement. ENSURE is lactose- andgluten-free, and is suitable for use in modified diets, includinglow-cholesterol diets. Although it is primarily an oral supplement, itcan be fed by tube.

[0325] Patient Conditions:

[0326] For patients on modified diets

[0327] For elderly patients at nutrition risk

[0328] For patients with involuntary weight loss

[0329] For patients recovering from illness or surgery

[0330] For patients who need a low-residue diet

[0331] Ingredients: -D Water, Sugar (Sucrose), Maltodextrin (Corn),Calcium and Sodium Caseinates, High-Oleic Safflower Oil, Soy ProteinIsolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium PhosphateTribasic, Sodium Citrate, Magnesium Chloride, Magnesium PhosphateDibasic, Artificial Flavor, Sodium Chloride, Soy Lecithin, CholineChloride, Ascorbic Acid, Carrageenan, Zinc Sulfate, Ferrous Sulfate,Alpha-Tocopheryl Acetate, Gellan Gum, Niacinamide, Calcium Pantothenate,Manganese Sulfate, Cupric Sulfate, Vitamin A Palmitate, ThiamineChloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, FolicAcid, Sodium Molybdate, Chromium Chloride, Biotin, Potassium Iodide,Sodium Selenate.

[0332] B. ENSURE® BARS:

[0333] Usage: ENSURE BARS are complete, balanced nutrition forsupplemental use between or with meals. They provide a delicious,nutrient-rich alternative to other snacks. ENSURE BARS contain <1 glactose/bar, and Chocolate Fudge Brownie flavor is gluten-free. (HoneyGraham Crunch flavor contains gluten.)

[0334] Patient Conditions:

[0335] For patients who need extra calories, protein, vitamins andminerals.

[0336] Especially useful for people who do not take in enough caloriesand nutrients.

[0337] For people who have the ability to chew and swallow

[0338] Not to be used by anyone with a peanut allergy or any type ofallergy to nuts.

[0339] Ingredients: Honey Graham Crunch—High-Fructose Corn Syrup, SoyProtein Isolate, Brown Sugar, Honey, Maltodextrin (Corn), Crisp Rice(Milled Rice,

[0340] Sugar [Sucrose], Salt [Sodium Chloride] and Malt), Oat Bran,Partially Hydrogenated Cottonseed and Soy Oils, Soy Polysaccharide,Glycerine, Whey Protein Concentrate, Polydextrose, Fructose, CalciumCaseinate, Cocoa Powder, Artificial Flavors, Canola Oil, High-OleicSafflower Oil, Nonfat Dry Milk, Whey Powder, Soy Lecithin and Corn Oil.Manufactured in a facility that processes nuts.

[0341] Vitamins and Minerals: Calcium Phosphate Tribasic, PotassiumPhosphate Dibasic, Magnesium Oxide, Salt (Sodium Chloride), PotassiumChloride, Ascorbic Acid, Ferric Orthophosphate, Alpha-TocopherylAcetate, Niacinamide, Zinc Oxide, Calcium Pantothenate, CopperGluconate, Manganese Sulfate, Riboflavin, Beta Carotene, PyridoxineHydrochloride, Thiamine Mononitrate, Folic Acid, Biotin, ChromiumChloride, Potassium Iodide, Sodium Selenate, Sodium Molybdate,Phylloquinone, Vitamin D3 and Cyanocobalamin.

[0342] Protein: Honey Graham Crunch—The protein source is a blend of soyprotein isolate and milk proteins. Soy protein isolate 74% Milk proteins26%

[0343] Fat: Honey Graham Crunch—The fat source is a blend of partiallyhydrogenated cottonseed and soybean, canola, high oleic safflower, oils,and soy lecithin. Partially hydrogenated cottonseed and soybean oil 76%Canola oil 8% High-oleic safflower oil 8% Corn oil 4% Soy lecithin 4%

[0344] Carbohydrate: Honey Graham Crunch—The carbohydrate source is acombination of high-fructose corn syrup, brown sugar, maltodextrin,honey, crisp rice, glycerine, soy polysaccharide, and oat bran.High-fructose corn syrup 24% Brown sugar 21% Maltodextrin 12% Honey 11%Crisp rice 9% Glycerine 9% Soy Polysaccharide 7% Oat bran 7%

[0345] C. ENSURE® HIGH PROTEIN:

[0346] Usage: ENSURE HIGH PROTEIN is a concentrated, high-protein liquidfood designed for people who require additional calories, protein,vitamins, and minerals in their diets. It can be used as an oralnutritional supplement with or between meals or, in appropriate amounts,as a meal replacement. ENSURE HIGH PROTEIN is lactose- and gluten-free,and is suitable for use by people recovering from general surgery or hipfractures and by patients at risk for pressure ulcers.

[0347] Patient Conditions:

[0348] For patients who require additional calories, protein, vitamins,and minerals, such as patients recovering from general surgery or hipfractures, patients at risk for pressure ulcers, and patients onlow-cholesterol diets.

[0349] Features:

[0350] Low in saturated fat

[0351] Contains 6 g of total fat and <5 mg of cholesterol per serving

[0352] Rich, creamy taste

[0353] Excellent source of protein, calcium, and other essentialvitamins and minerals

[0354] For low-cholesterol diets

[0355] Lactose-free, easily digested

[0356] Ingredients:

[0357] Vanilla Supreme: -D Water, Sugar (Sucrose), Maltodextrin (Corn),Calcium and Sodium Caseinates, High-OIeic Safflower Oil, Soy ProteinIsolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium PhosphateTribasic, Sodium Citrate, Magnesium Chloride, Magnesium PhosphateDibasic, Artificial Flavor, Sodium Chloride, Soy Lecithin, CholineChloride, Ascorbic Acid, Carrageenan, Zinc Sulfate, Ferrous Suffate,Alpha-Tocopheryl Acetate, Gellan Gum, Niacinamide, Calcium Pantothenate,Manganese Sulfate, Cupric Sulfate, Vitamin A Palmitate, ThiamineChloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, FolicAcid, Sodium Molybdate, Chromium Chloride, Biotin, Potassium Iodide,Sodium Selenate, Phylloquinone, Vitamin D3 and Cyanocobalamin.

[0358] Protein:

[0359] The protein source is a blend of two high-biologic-valueproteins: casein and soy. Sodium and calcium caseinates 85% Soy proteinisolate 15%

[0360] Fat:

[0361] The fat source is a blend of three oils: high-oleic safflower,canola, and soy. High-oleic safflower oil 40% Canola oil 30% Soy oil 30%

[0362]  The level of fat in ENSURE HIGH PROTEIN meets American HeartAssociation (AHA) guidelines. The 6 grams of fat in ENSURE HIGH PROTEINrepresent 24% of the total calories, with 2.6% of the fat being fromsaturated fatty acids and 7.9% from polyunsaturated fatty acids. Thesevalues are within the AHA guidelines of <30% of total calories from fat,<10% of the calories from saturated fatty acids, and <10% of totalcalories from polyunsaturated fatty acids.

[0363] Carbohydrate:

[0364] ENSURE HIGH PROTEIN contains a combination of maltodextrin andsucrose. The mild sweetness and flavor variety (vanilla supreme,chocolate royal, wild berry, and banana), plus VARI-FLAVORS® Flavor Pacsin pecan, cherry, strawberry, lemon, and orange, help to prevent flavorfatigue and aid in patient compliance.

[0365] Vanilla and other nonchocolate flavors: Sucrose 60% Maltodextrin40% Chocolate: Sucrose 70% Maltodextrin 30%

[0366] D. ENSURE® LIGHT

[0367] Usage: ENSURE LIGHT is a low-fat liquid food designed for use asan oral nutritional supplement with or between meals. ENSURE LIGHT islactose- and gluten-free, and is suitable for use in modified diets,including low-cholesterol diets.

[0368] Patient Conditions:

[0369] For normal-weight or overweight patients who need extra nutritionin a supplement that contains 50% less fat and 20% fewer calories thanENSURE.

[0370] For healthy adults who do not eat right and need extra nutrition.

[0371] Features:

[0372] Low in fat and saturated fat

[0373] Contains 3 g of total fat per serving and <5 mg cholesterol

[0374] Rich, creamy taste

[0375] Excellent source of calcium and other essential vitamins andminerals

[0376] For low-cholesterol diets

[0377] Lactose-free, easily digested

[0378] Ingredients:

[0379] French Vanilla: -D Water, Maltodextrin (Corn), Sugar (Sucrose),Calcium Caseinate, High-Oleic Safflower Oil, Canola Oil, MagnesiumChloride, Sodium Citrate, Potassium Citrate, Potassium PhosphateDibasic, Magnesium Phosphate Dibasic, Natural and Artificial Flavor,Calcium Phosphate Tribasic, Cellulose Gel, Choline Chloride, SoyLecithin, Carrageenan, Salt (Sodium Chloride), Ascorbic Acid, CelluloseGum, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Zinc Sulfate,Niacinamide, Manganese Sulfate, Calcium Pantothenate, Cupric Sulfate,Thiamine Chloride Hydrochloride, Vitamin A Palmitate, PyridoxineHydrochloride, Riboflavin, Chromium Chloride, Folic Acid, SodiumMolybdate, Biotin, Potassium Iodide, Sodium Selenate, Phylloquinone,Vitamin D3 and Cyanocobalamin.

[0380] Protein:

[0381] The protein source is calcium caseinate. Calcium caseinate 100%

[0382] Fat:

[0383] The fat source is a blend of two oils: high-oleic safflower andcanola. High-oleic safflower oil 70% Canola oil 30%

[0384]  The level of fat in ENSURE LIGHT meets American HeartAssociation (AHA) guidelines. The 3 grams of fat in ENSURE LIGHTrepresent 13.5% of the total calories, with 1.4% of the fat being fromsaturated fatty acids and 2.6% from polyunsaturated fatty acids. Thesevalues are within the AHA guidelines of <30% of total calories from fat,<10% of the, calories from saturated fatty acids, and <10% of totalcalories from polyunsaturated fatty acids.

[0385] Carbohydrate:

[0386] ENSURE LIGHT contains a combination of maltodextrin and sucrose.The chocolate flavor contains corn syrup as well. The mild sweetness andflavor variety (French vanilla, chocolate supreme, strawberry swirl),plus VARI-FLAVORS® Flavor Pacs in pecan, cherry, strawberry, lemon, andorange, help to prevent flavor fatigue and aid in patient compliance.

[0387] Vanilla and other nonchocolate flavors: Sucrose   51%Maltodextrin   49% Chocolate: Sucrose 47.0% Corn Syrup 26.5%Maltodextrin 26.5%

[0388] Vitamins and Minerals:

[0389] An 8-fl-oz serving of ENSURE LIGHT provides at least 25% of theRDIs for 24 key vitamins and minerals.

[0390] Caffeine:

[0391] Chocolate flavor contains 2.1 mg caffeine/8 fl oz.

[0392] E. ENSURE PLUS®

[0393] Usage: ENSURE PLUS is a high-calorie, low-residue liquid food foruse when extra calories and nutrients, but a normal concentration ofprotein, are needed. It is designed primarily as an oral nutritionalsupplement to be used with or between meals or, in appropriate amounts,as a meal replacement. ENSURE PLUS is lactose- and gluten-free. Althoughit is primarily an oral nutritional supplement, it can be fed by tube.

[0394] Patient Conditions:

[0395] For patients who require extra calories and nutrients, but anormal concentration of protein, in a limited volume.

[0396] For patients who need to gain or maintain healthy weight.

[0397] Features:

[0398] Rich, creamy taste

[0399] Good source of essential vitamins and minerals

[0400] Ingredients:

[0401] Vanilla: -D Water, Corn Syrup, Maltodextrin (Corn), Corn Oil,Sodium and Calcium Caseinates, Sugar (Sucrose), Soy Protein Isolate,Magnesium Chloride, Potassium Citrate, Calcium Phosphate Tribasic, SoyLecithin, Natural and Artificial Flavor, Sodium Citrate, PotassiumChloride, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc Sulfate,Ferrous Sulfate, Alpha-Tocopheryl Acetate, Niacinamide, CalciumPantothenate, Manganese Sulfate, Cupric Sulfate, Thiamine ChlorideHydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin APalmitate, Folic Acid, Biotin, Chromium Chloride, Sodium Molybdate,Potassium Iodide, Sodium Selenite, Phylloquinone, Cyanocobalamin andVitamin D3.

[0402] Protein:

[0403] The protein source is a blend of two high-biologic-valueproteins: casein and soy. Sodium and calcium caseinates 84% Soy proteinisolate 16%

[0404] Fat:

[0405] The fat source is corn oil. Corn oil 100%

[0406] Carbohydrate:

[0407] ENSURE PLUS contains a combination of maltodextrin and sucrose.The mild sweetness and flavor variety (vanilla, chocolate, strawberry,coffee, buffer pecan, and eggnog), plus VARI-FLAVORS® Flavor Pacs inpecan, cherry, strawberry, lemon, and orange, help to prevent flavorfatigue and aid in patient compliance.

[0408] Vanilla, strawberry, butter pecan, and coffee flavors: Corn Syrup39% Maltodextrin 38% Sucrose 23%

[0409] Chocolate and eggnog flavors: Corn Syrup 36% Maltodextrin 34%Sucrose 30%

[0410] Vitamins and Minerals:

[0411] An 8-fl-oz serving of ENSURE PLUS provides at least 15% of theRDIs for 25 key Vitamins and minerals.

[0412] Caffeine:

[0413] Chocolate flavor contains 3.1 mg Caffeine/8 fl oz. Coffee flavorcontains a trace amount of caffeine.

[0414] F. ENSURE PLUS® HN

[0415] Usage: ENSURE PLUS HN is a nutritionally complete high-calorie,high-nitrogen liquid food designed for people with higher calorie andprotein needs or limited volume tolerance. It may be used for oralsupplementation or for total nutritional support by tube. ENSURE PLUS HNis lactose- and gluten-free.

[0416] Patient Conditions:

[0417] For patients with increased calorie and protein needs, such asfollowing surgery or injury.

[0418] For patients with limited volume tolerance and early satiety.

[0419] Features:

[0420] For supplemental or total nutrition

[0421] For oral or tube feeding

[0422] 1.5 CaVmL,

[0423] High nitrogen

[0424] Calorically dense

[0425] Ingredients:

[0426] Vanilla: -D Water, Maltodextrin (Corn), Sodium and CalciumCaseinates, Corn Oil, Sugar (Sucrose), Soy Protein Isolate, MagnesiumChloride, Potassium Citrate, Calcium Phosphate Tribasic, Soy Lecithin,Natural and Artificial Flavor, Sodium Citrate, Choline Chloride,Ascorbic Acid, Taurine, L-Carnitine, Zinc Sulfate, Ferrous Sulfate,Alpha-Tocopheryl Acetate, Niacinamide, Carrageenan, CalciumPantothenate, Manganese Sulfate, Cupric Sulfate, Thiamine ChlorideHydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin APalmitate, Folic Acid, Biotin, Chromium Chloride, Sodium Molybdate,Potassium Iodide, Sodium Selenite, Phylloquinone, Cyanocobalamin andVitamin D3.

[0427] G. ENSURE® POWDER:

[0428] Usage: ENSURE POWDER (reconstituted with water) is a low-residueliquid food designed primarily as an oral nutritional supplement to beused with or between meals. ENSURE POWDER is lactose- and gluten-free,and is suitable for use in modified diets, including low-cholesteroldiets.

[0429] Patient Conditions:

[0430] For patients on modified diets

[0431] For elderly patients at nutrition risk

[0432] For patients recovering from illness/surgery

[0433] For patients who need a low-residue diet

[0434] Features:

[0435] Convenient, easy to mix

[0436] Low in saturated fat

[0437] Contains 9 g of total fat and <5 mg of cholesterol per serving

[0438] High in vitamins and minerals

[0439] For low-cholesterol diets

[0440] Lactose-free, easily digested

[0441] Ingredients: -D Corn Syrup, Maltodextrin (Corn), Sugar (Sucrose),Corn Oil, Sodium and Calcium Caseinates, Soy Protein Isolate, ArtificialFlavor, Potassium Citrate, Magnesium Chloride, Sodium Citrate, CalciumPhosphate Tribasic, Potassium Chloride, Soy Lecithin, Ascorbic Acid,Choline Chloride, Zinc Sulfate, Ferrous Sulfate, Alpha-TocopherylAcetate, Niacinamide, Calcium Pantothenate, Manganese Sulfate, ThiamineChloride Hydrochloride, Cupric Sulfate, Pyridoxine Hydrochloride,Riboflavin, Vitamin A Palmitate, Folic Acid, Biotin, Sodium Molybdate,Chromium Chloride, Potassium Iodide, Sodium Selenate, Phylloquinone,Vitamin D3 and Cyanocobalamin.

[0442] Protein:

[0443] The protein source is a blend of two high-biologic-valueproteins: casein and soy. Sodium and calcium caseinates 84% Soy proteinisolate 16%

[0444] Fat:

[0445] The fat source is corn oil. Corn oil 100%

[0446] Carbohydrate:

[0447] ENSURE POWDER contains a combination of corn syrup, maltodextrin,and sucrose. The mild sweetness of ENSURE POWDER, plus VARI-FLAVORS®Flavor Pacs in pecan, cherry, strawberry, lemon, and orange, helps toprevent flavor fatigue and aid in patient compliance.

[0448] Vanilla: Corn Syrup 35% Maltodextrin 35% Sucrose 30%

[0449] H. ENSURE® PUDDING

[0450] Usage: ENSURE PUDDING is a nutrient-dense supplement providingbalanced nutrition in a nonliquid form to be used with or between meals.It is appropriate for consistency-modified diets (e.g., soft, pureed, orfull liquid) or for people with swallowing impairments. ENSURE PUDDINGis gluten-free.

[0451] Patient Conditions:

[0452] For patients on consistency-modified diets (e.g., soft, pureed,or full liquid)

[0453] For patients with swallowing impairments

[0454] Features:

[0455] Rich and creamy, good taste

[0456] Good source of essential vitamins and minerals

[0457] Convenient-needs no refrigeration

[0458] Gluten-free

[0459] Nutrient Profile per 5 oz: Calories 250, Protein 10.9%, Total Fat34.9%, Carbohydrate 54.2%

[0460] Ingredients:

[0461] Vanilla: -D Nonfat Milk, Water, Sugar (Sucrose), PartiallyHydrogenated Soybean Oil, Modified Food Starch, Magnesium Sulfate,Sodium Stearoyl Lactylate, Sodium Phosphate Dibasic, Artificial Flavor,Ascorbic Acid, Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate,Choline Chloride, Niacinamide, Manganese Sulfate, Calcium Pantothenate,FD&C Yellow #5, Potassium Citrate, Cupric Sulfate, Vitamin A Palmitate,Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin,FD&C Yellow #6, Folic Acid, Biotin, Phylloquinone, Vitamin D3 andCyanocobalamin.

[0462] Protein:

[0463] The protein source is nonfat milk. Nonfat milk 100%

[0464] Fat:

[0465] The fat source is hydrogenated soybean oil. Hydrogenated soybeanoil 100%

[0466] Carbohydrate:

[0467] ENSURE PUDDING contains a combination of sucrose and modifiedfood starch. The mild sweetness and flavor variety (vanilla, chocolate,butterscotch, and tapioca) help prevent flavor fatigue. The productcontains 9.2 grams of lactose per serving.

[0468] Vanilla and other nonchocolate flavors: Sucrose 56% Lactose 27%Modified food starch 17%

[0469] Chocolate: Sucrose 58% Lactose 26% Modified food starch 16%

[0470] I. ENSURE® WITH FIBER:

[0471] Usage: ENSURE WITH FIBER is a fiber-containing, nutritionallycomplete liquid food designed for people who can benefit from increaseddietary fiber and nutrients. ENSURE WITH FIBER is suitable for peoplewho do not require a low-residue diet. It can be fed orally or by tube,and can be used as a nutritional supplement to a regular diet or, inappropriate amounts, as a meal replacement. ENSURE WITH FIBER islactose- and gluten-free, and is suitable for use in modified diets,including low-cholesterol diets.

[0472] Patient Conditions:

[0473] For patients who can benefit from increased dietary fiber andnutrients

[0474] Features:

[0475] New advanced formula-low in saturated fat, higher in vitamins andminerals

[0476] Contains 6 g of total fat and <5 mg of cholesterol per serving

[0477] Rich, creamy taste

[0478] Good source of fiber

[0479] Excellent source of essential vitamins and minerals

[0480] For low-cholesterol diets

[0481] Lactose- and gluten-free

[0482] Ingredients:

[0483] Vanilla: -D Water; Maltodextrin (Corn), Sugar (Sucrose), Sodiumand Calcium Caseinates, Oat Fiber, High-Oleic Safflower Oil, Canola Oil,Soy Protein Isolate, Corn Oil, Soy Fiber, Calcium Phosphate Tribasic,Magnesium Chloride, Potassium Citrate, Cellulose Gel, Soy Lecithin,Potassium Phosphate Dibasic, Sodium Citrate, Natural and ArtificialFlavors, Choline Chloride, Magnesium Phosphate, Ascorbic Acid, CelluloseGum, Potassium Chloride, Carrageenan, Ferrous Sulfate, Alpha-TocopherylAcetate, Zinc Sulfate, Niacinamide, Manganese Sulfate, CalciumPantothenate, Cupric Sulfate, Vitamin A Palmitate, Thiamine ChlorideHydrochloride, Pyridoxine Hydrochloride, Riboflavin, Folic Acid,Chromium Chloride, Biotin, Sodium Molybdate, Potassium Iodide, SodiumSelenate, Phylloquinone, Vitamin D3 and Cyanocobalamin.

[0484] Protein:

[0485] The protein source is a blend of two high-biologic-valueproteins-casein and soy. Sodium and calcium caseinates 80% Soy proteinisolate 20%

[0486] Fat:

[0487] The fat source is a blend of three oils: high-oleic safflower,canola, and corn. High-oleic safflower oil 40% Canola oil 40% Corn oil20%

[0488]  The level of fat in ENSURE WITH FIBER meets American HeartAssociation (AHA) guidelines. The 6 grams of fat in ENSURE WITH FIBERrepresent 22% of the total calories, with 2.01% of the fat being fromsaturated fatty acids and 6.7% from polyunsaturated fatty acids. Thesevalues are within the AHA guidelines of ≦30% of total calories from fat,<10% of the calories from saturated fatty acids, and ≦10% of totalcalories from polyunsaturated fatty acids.

[0489] Carbohydrate:

[0490] ENSURE WITH FIBER contains a combination of maltodextrin andsucrose. The mild sweetness and flavor variety (vanilla, chocolate, andbutter pecan), plus VARI-FLAVORS® Flavor Pacs in pecan, cherry,strawberry, lemon, and orange, help to prevent flavor fatigue and aid inpatient compliance.

[0491] Vanilla and other nonchocolate flavors: Maltodextrin 66% Sucrose25% Oat Fiber 7% Soy Fiber 2%

[0492] Chocolate: Maltodextrin 55% Sucrose 36% Oat Fiber 7% Soy Fiber 2%

[0493] Fiber:

[0494] The fiber blend used in ENSURE WITH FIBER consists of oat fiberand soy polysaccharide. This blend results in approximately 4 grams oftotal dietary fiber per 8-fl. oz can. The ratio of insoluble to solublefiber is 95:5.

[0495] The various nutritional supplements described above and known toothers of skill in the art can be substituted and/or supplemented withthe PUFAs produced in accordance with the present invention.

[0496] J. Oxepa™ Nutritional Product

[0497] Oxepa is a low-carbohydrate, calorically dense, enteralnutritional product designed for the dietary management of patients withor at risk for ARDS. It has a unique combination of ingredients,including a patented oil blend containing eicosapentaenoic acid (EPAfrom fish oil), γ-linolenic acid (GLA from borage oil), and elevatedantioxidant levels.

[0498] Caloric Distribution:

[0499] Caloric density is high at 1.5 Cal/mL (355 Cal/8 fl oz), tominimize the volume required to meet energy needs. The distribution ofCalories in Oxepa is shown in Table A. TABLE A Caloric Distribution ofOxepa per 8 fl oz. per liter % of Cal Calories 355 1,500 — Fat (g) 22.293.7 55.2 Carbohydrate (g) 25 105.5 28.1 Protein (g) 14.8 62.5 16.7Water (g) 186 785 —

[0500] Fat:

[0501] Oxepa contains 22.2 g of fat per 8-fl oz serving (93.7 g/L).

[0502] The fat source is an oil blend of 31.8% canola oil, 25%medium-chain triglycerides (MCTs), 20% borage oil, 20% fish oil, and3.2% soy lecithin. The typical fatty acid profile of Oxepa is shown inTable B.

[0503] Oxepa provides a balanced amount of polyunsaturated,monounsaturated, and saturated fatty acids, as shown in Table VI.

[0504] Medium-chain trigylcerides (MCTs)—25% of the fat blend—aidgastric emptying because they are absorbed by the intestinal tractwithout emulsification by bile acids.

[0505] The various fatty acid components of Oxepa™ nutritional productcan be substituted and/or supplemented with the PUFAs produced inaccordance with this invention. TABLE B Typical Fatty Acid Profile FattyAcids % Total g/8 fl oz* 9/L* Caproic (6:0) 0.2 0.04 0.18 Caprylic (8:0)14.69 3.1 13.07 Capric (10:0) 11.06 2.33 9.87 Palmitic (16:0) 5.59 1.184.98 Palmitoleic 1.82 0.38 1.62 Stearic 1.94 0.39 1.64 Oleic 24.44 5.1621.75 Linoleic 16.28 3.44 14.49 α-Linolenic 3.47 0.73 3.09 γ-Linolenic4.82 1.02 4.29 Eicosapentaenoic 5.11 1.08 4.55 n-3-Docosapent-aenoic0.55 0.12 0.49 Docosahexaenoic 2.27 0.48 2.02 Others 7.55 1.52 6.72

[0506] TABLE C Fat Profile of Oxepa % of total calories from fat 55.2Polyunsaturated fatty acids 31.44 g/L Monounsaturated fatty acids 25.53g/L Saturated fatty acids 32.38 g/L n-6 to n-3 ratio 1.75:1 Cholesterol9.49 mg/8 fl oz 40.1 mg/L

[0507] Carbohydrate:

[0508] The carbohydrate content is 25.0 g per 8-fl-oz serving (105.5g/L).

[0509] The carbohydrate sources are 45% maltodextrin (a complexcarbohydrate) and 55% sucrose (a simple sugar), both of which arereadily digested and absorbed.

[0510] The high-fat and low-carbohydrate content of Oxepa is designed tominimize carbon dioxide (CO2) production. High CO2 levels can complicateweaning in ventilator-dependent patients. The low level of carbohydratealso may be useful for those patients who have developed stress-inducedhyperglycemia.

[0511] Oxepa is lactose-free.

[0512] Dietary carbohydrate, the amino acids from protein, and theglycerol moiety of fats can be converted to glucose within the body.Throughout this process, the carbohydrate requirements ofglucose-dependent tissues (such as the central nervous system and redblood cells) are met. However, a diet free of carbohydrates can lead toketosis, excessive catabolism of tissue protein, and loss of fluid andelectrolytes. These effects can be prevented by daily ingestion of 50 to100 g of digestible carbohydrate, if caloric intake is adequate. Thecarbohydrate level in Oxepa is also sufficient to minimizegluconeogenesis, if energy needs are being met.

[0513] Protein:

[0514] Oxepa contains 14.8 g of protein per 8-fl-oz serving (62.5 g/L).

[0515] The total calorie/nitrogen ratio (150:1) meets the need ofstressed patients.

[0516] Oxepa provides enough protein to promote anabolism and themaintenance of lean body mass without precipitating respiratoryproblems. High protein intakes are a concern in patients withrespiratory insufficiency. Although protein has little effect on CO2production, a high protein diet will increase ventilatory drive.

[0517] The protein sources of Oxepa are 86.8% sodium caseinate and 13.2%calcium caseinate.

[0518] The amino acid profile of the protein system in Oxepa meets orsurpasses the standard for high quality protein set by the NationalAcademy of Sciences.

[0519] Oxepa is gluten-free.

What is claimed is:
 1. An isolated nucleotide acid sequence or fragmentthereof comprising or complementary to a nucleotide sequence encoding apolypeptide having desaturase activity, wherein the amino acid sequenceof said polypeptide has at least 50% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:26 and SEQ IDNO:42.
 2. An isolated nucleotide sequence or fragment thereof comprisingor complementary to at least 50% of the nucleotide sequence selectedfrom the group consisting of SEQ ID NO:25 and SEQ ID NO:41.
 3. Theisolated nucleotide sequence of claim 2, wherein said sequence isselected from the group consisting of SEQ ID NO:25 and SEQ ID NO:41. 4.The isolated nucleotide sequence of claim 2 or 3, wherein said sequenceencodes a functionally active desaturase which utilizes apolyunsaturated fatty acid as a substrate.
 5. The isolated nucleotidesequence of claim 1 or 2, wherein said sequence is derived fromSaprolegnia diclina.
 6. A purified polypeptide encoded by said isolatednucleotide sequence of claim 1 or
 2. 7. A purified polypeptide thatdesaturates a polyunsaturated fatty acid substrate at an omega-3 carbonof said substrate and has at least 50% amino acid identity to an aminoacid sequence comprising SEQ ID NO:
 26. 8. The purified polypeptide ofclaim 7, wherein said polypeptide desaturates a fatty acid substratehaving 20 carbon atoms.
 9. A purified polypeptide that desaturates apolyunsaturated fatty acid substrate at a delta-12 carbon of saidsubstrate and has at least 50% amino acid identity to SEQ ID NO:
 42. 10.The purified polypeptide of claim 9, wherein said polypeptidedesaturates a fatty acid substrate having 18 carbon atoms.
 11. A methodof producing a desaturase comprising the steps of: (a) isolating anucleotide sequence comprising or complementary to at least 50% of thenucleotide sequence selected from the group consisting of SEQ ID NO: 25and SEQ ID NO: 41; (b) constructing a vector comprising said isolatednucleotide sequence of step (a); and (c) introducing said vector of step(b) into a host cell for a time and under conditions sufficient forexpression of a desaturase encoded by said isolated nucleotide sequenceof step (a).
 12. A vector comprising: 1) an isolated nucleotide sequencecorresponding to or complementary to at least 50% of the nucleotidesequence selected from the group consisting of SEQ ID NO: 25 and SEQ IDNO: 41, operably linked to b) a regulatory sequence.
 13. A host cellcomprising said vector of claim
 12. 14. The host cell of claim 1,wherein said host cell is a eukaryotic cell selected from the groupconsisting of a mammalian cell, an insect cell, a plant cell and afungal cell.
 15. The host cell of claim 14, wherein expression of saidisolated nucleotide sequence of said vector results in said host cellproducing a polyunsaturated fatty acid that is not produced in awild-type of said host cell.
 16. A plant cell, plant, or plant tissuecomprising said vector of claim 12, wherein expression of saidnucleotide sequence of said vector results in production of apolyunsaturated fatty acid by said plant cell, plant or plant tissue.17. The plant cell, plant, or plant tissue of claim 16, wherein saidvector induces the production of a polyunsaturated fatty acid selectedfrom the group consisting of linoleic acid, eicosatetraenoic acid andeicosapentaenoic acid.
 18. A transgenic plant comprising said vector ofclaim 12, wherein expression of said nucleotide sequence of said vectorresults in production of a polyunsaturated fatty acid in seeds of saidtransgenic plant.
 19. One or more plant oils or acids expressed by saidplant cell, plant or plant tissue of claim
 16. 20. A method forproducing a polyunsaturated fatty acid comprising the steps of: (a)isolating a nucleotide sequence comprising or complementary to at least50% of the nucleotide sequence selected from the group consisting of SEQID NO: 25 and SEQ ID NO: 41; (b) constructing a vector comprising saidisolated nucleotide sequence of step (a); (c) transforming the vector ofstep (b) into a host cell for a time and under conditions sufficient forexpression of a desaturase encoded by said isolated nucleotide sequenceof step (a); and (d) exposing said expressed desaturase selected fromthe group consisting of an omega-3-desaturase and a delta 12-desaturase,to a fatty acid substrate, whereby said substrate is catalyticallyconverted by said desaturase into a desired polyunsaturated fatty acidproduct.
 21. The method of claim 20, wherein said substrate isdihomo-gamma-linolenic acid or arachidonic acid and said productpolyunsaturated fatty acid is eicosatetraenoic acid or eicosapentaenoicacid, respectively, when said expressed desaturase is anomega-3-desaturase.
 22. The method of claim 20, wherein said substratepolyunsaturated fatty acid is oleic acid and said productpolyunsaturated fatty acid is linoleic acid, when said expresseddesaturase is a delta 12-desaturase.
 23. The method of claim 20, furthercomprising, after step (d), the step of: (e) exposing saidpolyunsaturated fatty acid product of step (d) to one or more enzymesselected from the group consisting of a desaturase and an elongase,whereby the polyunsaturated fatty acid product of step (d) iscatalytically converted into another polyunsaturated fatty acid product.24. The method of claim 23, wherein said product polyunsaturated fattyacid is eicosatetraenoic acid or eicosapentaenoic acid and said anotherpolyunsaturated fatty acid is eicosapentaenoic acid or omega3-docosapentaenoic acid, respectively, when said expressed desaturase ofstep (d) is an omega 3-desaturase.
 25. The method of claim 23, whereinsaid product polyunsaturated fatty acid is linoleic acid and saidanother polyunsaturated fatty acid is gamma-linolenic acid, when saidexpressed desaturase of step (d) is a delta 12-desaturase.
 26. Themethod of claim 23 further comprising the step of exposing said anotherpolyunsaturated fatty acid to one or more enzymes selected from thegroup consisting of a desaturase and an elongase in order to convertsaid another polyunsaturated fatty acid to a final polyunsaturated fattyacid.
 27. The method of claim 26 wherein said final polyunsaturatedfatty acid is selected from the group consisting of omega3-docosapentaenoic acid and docosahexaenoic acid, when said expresseddesaturase of step (d) is an omega 3-desaturase.
 28. The method of claim26 wherein said final polyunsaturated fatty acid is selected from thegroup consisting of dihomo-gamma-linolenic acid, arachidonic acid,adrenic acid, omega 6-docosapentaenoic acid, eicosatetraenoic acid,stearidonic acid, eicosapentaenoic acid, omega 3-docosapentaenoic acidand docosahexaenoic acid, when said expressed desaturase of step (d) isa delta 12-desaturase.
 29. A method of producing a polyunsaturated fattyacid comprising exposing a fatty acid substrate to a polypeptide havingat least 50% amino acid identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 26 and SEQ ID NO: 42, whereby saidfatty acid substrate is catalytically converted into saidpolyunsaturated fatty acid.
 30. The method of claim 29, wherein thefatty acid substrate is dihomo-gamma-linolenic acid or arachidonic acidand said product polyunsaturated fatty acid is eicosatetraenoic acid oreicosapentaenoic acid, respectively, when said polypeptide is an omega3-desaturase.
 31. The method of claim 29, wherein said fatty acidsubstrate is oleic acid and said polyunsaturated fatty acid is linoleicacid, when said polypeptide is a delta 12-desaturase.
 32. A compositioncomprising at least one polyunsaturated fatty acid selected from thegroup consisting of said product polyunsaturated fatty acid producedaccording to the method of claim 20, said another polyunsaturated fattyacid produced according to the method of claim 23, and said finalpolyunsaturated fatty acid produced according to the method of claim 26.33. The composition of claim 32, wherein said product polyunsaturatedfatty acid is eicosatetraenoic acid or eicosapentaenoic acid, when saidexpressed desaturase of step (d) is an omega 3-desaturase.
 34. Thecomposition of claim 32, wherein said product polyunsaturated fatty acidis linoleic acid, when said expressed desaturase of step (d) is a delta12-desaturase.
 35. The composition of claim 32, wherein said anotherpolyunsaturated fatty acid is eicosapentaenoic acid or omega3-docosapentaenoic acid, respectively, when said expressed desaturase ofstep (d) is an omega 3-desaturase.
 36. The composition of claim 32,wherein said another polyunsaturated fatty acid is gamma-linolenic acid,when said expressed desaturase of step (d) is a delta 12-desaturase. 37.The composition of claim 32, wherein said final polyunsaturated fattyacid is selected from the group consisting of omega 3-docosapentaenoicacid and docosahexaenoic acid, when said expressed desaturase of step(d) is an omega 3-desaturase.
 38. The composition of claim 32, whereinsaid final polyunsaturated fatty acid is selected from the groupconsisting of dihomo-gamma-linolenic acid, arachidonic acid, adrenicacid, omega 6-docosapentaenoic acid, eicosatetraenoic acid, stearidonicacid, eicosapentaenoic acid, omega 3-docosapentaenoic acid anddocosahexaenoic acid, when said expressed desaturase of step (d) is adelta 12-desaturase.
 39. A method of preventing or treating a conditioncaused by insufficient intake of at least one polyunsaturated fatty acidcomprising administering to said patient said composition of claim 32 inan amount sufficient to effect said prevention or treatment.